Immunostimulatory nucleic acids

ABSTRACT

The invention relates to a class of soft or semi-soft CpG immunostimulatory oligonucleotides that are useful for stimulating an immune response.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 60/404,479, filed Aug. 19, 2002, U.S.Ser. No. 60/404,820 filed Aug. 19, 2002, U.S. Ser. No. 60/429,701 filedNov. 27, 2002, and U.S. Ser. No. 60/447,377 filed Feb. 14, 2003 whichare herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to immunostimulatory nucleicacids, as well as immunostimulatory oligonucleotides with reduced renalinflammatory effects, compositions thereof and methods of using theimmunostimulatory nucleic acids.

BACKGROUND OF THE INVENTION

Bacterial DNA has immune stimulatory effects to activate B cells andnatural killer cells, but vertebrate DNA does not (Tokunaga, T., et al.,1988. Jpn. J. Cancer Res. 79:682-686; Tokunaga, T., et al., 1984, JNCI72:955-962; Messina, J. P., et al., 1991, J. Immunol. 147:1759-1764; andreviewed in Krieg, 1998, In: Applied Oligonucleotide Technology, C. A.Stein and A. M. Krieg, (Eds.), John Wiley and Sons, Inc., New York,N.Y., pp. 431-448). It is now understood that these immune stimulatoryeffects of bacterial DNA are a result of the presence of unmethylatedCpG dinucleotides in particular base contexts (CpG motifs), which arecommon in bacterial DNA, but methylated and underrepresented invertebrate DNA (Krieg et al, 1995 Nature 374:546-549; Krieg, 1999Biochim. Biophys. Acta 93321:1-10). The immune stimulatory effects ofbacterial DNA can be mimicked with synthetic oligodeoxynucleotides (ODN)containing these CpG motifs. Such CpG ODN have highly stimulatoryeffects on human and murine leukocytes, inducing B cell proliferation;cytokine and immunoglobulin secretion; natural killer (NK) cell lyticactivity and IFN-γ secretion; and activation of dendritic cells (DCs)and other antigen presenting cells to express costimulatory moleculesand secrete cytokines, especially the Th1-like cytokines that areimportant in promoting the development of Th1-like T cell responses.These immune stimulatory effects of native phosphodiester backbone CpGODN are highly CpG specific in that the effects are dramatically reducedif the CpG motif is methylated, changed to a GpC, or otherwiseeliminated or altered (Krieg et al, 1995 Nature 374:546-549; Hartmann etal, 1999 Proc. Natl. Acad. Sci USA 96:9305-10).

In early studies, it was thought that the immune stimulatory CpG motiffollowed the formula purine-purine-CpG-pyrimidine-pyrimidine (Krieg etal, 1995 Nature 374:546-549; Pisetsky, 1996 J. Immunol. 156:421-423;Hacker et al., 1998 EMBO J. 17:6230-6240; Lipford et al, 1998 Trends inMicrobiol. 6:496-500). However, it is now clear that mouse lymphocytesrespond quite well to phosphodiester CpG motifs that do not follow this“formula” (Yi et al., 1998 J. Immunol. 160:5898-5906) and the same istrue of human B cells and dendritic cells (Hartmann et al, 1999 Proc.Natl. Acad. Sci USA 96:9305-10; Liang, 1996 J. Clin. Invest.98:1119-1129).

Several different classes of CpG nucleic acids has recently beendescribed. One class is potent for activating B cells but is relativelyweak in inducing IFN-α and NK cell activation; this class has beentermed the B class. The B class CpG nucleic acids typically are fullystabilized and include an unmethylated CpG dinucleotide within certainpreferred base contexts. See, e.g., U.S. Pat. Nos. 6,194,388; 6,207,646;6,214,806; 6,218,371; 6,239,116; and 6,339,068. Another class of CpGnucleic acids activates B cells and NK cells and induces IFN-α; thisclass has been termed the C-class. The C-class CpG nucleic acids, asfirst characterized, typically are fully stabilized, include a Bclass-type sequence and a GC-rich palindrome or near-palindrome. Thisclass has been described in co-pending U.S. provisional patentapplication 60/313,273, filed Aug. 17, 2001 and U.S. Ser. No. 10/224,523filed on Aug. 19, 2002 and related PCT Patent Application PCT/US02/26468published under International Publication Number WO 03/015711.

SUMMARY OF THE INVENTION

It has been surprisingly discovered that immunostimulatory properties ofthe B-class and C-class CpG nucleic acids and other stabilizedimmunostimulatory nucleic acids can be maintained or even improved bythe selective inclusion of one or more non-stabilized linkages betweencertain nucleotides. The non-stabilized linkages are preferably naturallinkages, i.e., phosphodiester linkages or phosphodiester-like linkages.A non-stabilized linkage will typically, but not necessarily, berelatively susceptible to nuclease digestion. The immunostimulatorynucleic acids of the instant invention include at least onenon-stabilized linkage situated between a 5′ pyrimidine (Y) and anadjacent 3′ purine (Z), preferably a guanine (G), wherein both the 5′ Yand the 3′ Z are internal nucleotides.

Like fully stabilized immunostimulatory nucleic acids, theimmunostimulatory nucleic acids of the instant invention are useful forinducing a Th1-like immune response. Accordingly, the immunostimulatorynucleic acids of the instant invention are useful as adjuvants forvaccination, and they are useful for treating diseases including cancer,infectious disease, allergy, and asthma. They are believed to be ofparticular use in any condition calling for prolonged or repeatedadministration of immunostimulatory nucleic acid for any purpose.

In addition to being useful for any purpose for which fully stabilizedimmunostimulatory nucleic acids have utility, the immunostimulatorynucleic acids of the instant invention may in some embodiments haveadvantages over fully stabilized immunostimulatory nucleic acids, suchas, increased potency and decreased toxicity.

The present invention relates in part to immunostimulatory CpGcontaining oligonucleotides. In one aspect the invention is anoligonucleotide having the formula: 5′T*C*G*T*CGTTTTGAN₁CGN₂*T*T3′ (SEQID NO:296). In the oligonucleotide N₁ is 0-6 nucleotides and N₂ is 0-7nucleotides. The symbol * refers to the presence of a stabilizedinternucleotide linkage. Internucleotide linkages not marked with an *may be unstabilized or stabilized, as long as the oligonucleotideincludes at least 2 phosphodiester internucleotide linkages. Thestabilized internucleotide linkage may be a phosphorothioate linkage. Insome embodiments N₁ is 0-2 nucleotides. Preferably the oligonucleotideis 16-24 nucleotides in length.

In some embodiment the oligonucleotide has one of the followingstructures:

(SEQ ID NO: 296) 5′T*C*G*T*C*G*TTTTGAN₁C*G*N₂*T*T3′, (SEQ ID NO: 296)5′T*C*G*T*C*G*T*T_T_T_GAN₁C*G*N₂*T*T3′ or (SEQ ID NO: 296)5′T*C*G*T*C*G*T*T*T*T*GA_N₁C*G*N₂*T*T3′The symbol _ refers to the presence of a phosphodiester internucleotidelinkage.

Preferably the oligonucleotide is

5′T*C*G*T*C*G*T*T*T*T*G*A_C_C_G_G_T*T*C*G*T*G*T*T3′, (SEQ ID NO: 297)5′T*C*G*T*C*G*T*T*T*T*G_A_C*G*T*T*T*T*G*T*C*G*T*T3′, (SEQ ID NO: 298)5′T*C*G*T*C*G*T*T_T_T_G*A*C*G*T*T*T*T3′, (SEQ ID NO: 299) or5′T*C*G*T*C*G*T*T_T_T_G*A*C*G*T*T3′. (SEQ ID NO: 300)

The invention, in other aspects, relates to an oligonucleotidecomprising: 5′T*C*G*(T*/A*)TN₃CGTTTTN₄CGN₅*T*T 3′ (SEQ ID NO:301). N₃ is0-4 nucleotides. N₄ is 1-5 nucleotides. N₅ is 0-7 nucleotides. Thesymbol * refers to the presence of a stabilized internucleotide linkage.Internucleotide linkages not marked with an * may be unstabilized orstabilized, as long as the oligonucleotide includes at least 2phosphodiester internucleotide linkages. The stabilized internucleotidelinkage may be a phosphorothioate linkage. In some embodiments N₄ is 1-2nucleotides. Preferably the oligonucleotide is 16-24 nucleotides inlength.

In some embodiment the oligonucleotide has one of the followingstructures: 5′

(SEQ ID NO: 301) 5′T*C*G*(T*/A*)TN₃CGTTTTN₄C*G*N₅*T*T 3′,(SEQ ID NO: 302) 5′T*C*G*A*T*N₃C*G*TTTTN₄C_G_*N₅*T*T 3′, or(SEQ ID NO: 303) 5′T*C*G*T*T*N₃C_G_TTTTN₄CGN₅*T*T 3′.

Preferably the oligonucleotide is

5′T*C*G*A*T*C*G*T*T*T*T_T_C_G*T*G*C*G*T*T*T*T*T3, (SEQ ID NO: 304) or5′T*C*G*T*T*T*T*G*A_C_G_T*T*T*T*G*T*C*G*T*T3′. (SEQ ID NO: 305)

According to other aspects, an oligonucleotide comprising:5′T*C*G*T*C*GNNNCGNCGNNNC*G*N*C*G*T*T3′ (SEQ ID NO:306) is provided. Nis any nucleotide. The symbol * refers to the presence of a stabilizedinternucleotide linkage. Internucleotide linkages not marked with an *may be unstabilized or stabilized, as long as the oligonucleotideincludes at least 3 phosphodiester internucleotide linkages. Thestabilized internucleotide linkage may be a phosphorothioate linkage. Insome embodiments the oligonucleotide includes 5 phosphodiesterinternucleotide linkages. Preferably the oligonucleotide is 16-24nucleotides in length.

In some embodiment the oligonucleotide has one of the followingstructures:

5′T*C*G*T*C*G*N*N*N*C_G_N_C_G_N*N*N*C*G*N*C*G*T*T 3′, (SEQ ID NO: 307)5′T*C*G*T*C*G*T*T*A*C_G_N_C_G_T*T*A*C*G*N*C*G*T*T 3′, (SEQ ID NO: 308)or 5′T*C*G*T*C*G*N*N*N*C_G_T_C_G_N*N*N*C*G*T*C*G*T*T 3′.(SEQ ID NO: 309)In one embodiment the oligonucleotide is5′T*C*G*T*C*G*T*T*A*C_G_T_C_G_T*T*A*C*G*T*C*G*T*T 3′ (SEQ ID NO:310).The symbol _ refers to the presence of a phosphodiester internucleotidelinkage.

In other embodiments the oligonucleotide includes at least one C_G motifwith a phosphodiester internucleotide linkage. In yet other embodimentsthe oligonucleotide does not include any C_G motifs with aphosphodiester internucleotide linkage.

In other aspects an oligonucleotide having the structure 5′T*C_G(N₆C_GN₇)₂₋₃T*C_G*T*T3′ (SEQ ID NOS:311-312) is provided. N₆ and N₇ areindependently between 1 and 5 nucleotides in length and theoligonucleotide has a length of 16-40 nucleotides.

In some embodiments N₆ is one nucleotide, for instance N₆ may be T or A.N₇ in some embodiments is five nucleotides, for example, N₇ may be fivepyrimidines or TTTTG.

In some embodiments the oligonucleotide has the structure:

5′ T*C_G*T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C_G*T*T 3′ (SEQ ID NO: 313)or 5′ T*C_G*A*C_G*T*T*T*T*G*T*C_G*T*T*T*T*G*T*C_G*T*T 3′.(SEQ ID NO: 314)

An oligonucleotide having the structure 5′T*CGCGN₈CGCGC*GN₉3′ (SEQ IDNO:315) is provided according to other aspects of the invention. N₈ isbetween 4 and 10 nucleotides in length and includes at least 1 C_Gmotif. N₉ is between 0 and 3 nucleotides in length. The oligonucleotidehas a length of 15-40 nucleotides.

In some embodiments N₈ includes at least 2 or 3 CG motifs. In otherembodiments N₈ is PuCGPyPyCG or PuCGPyPyCGCG. Optionally N₈ is ACGTTCG.N₉ may include at least on CG motif, such as, CCG.

In some embodiments the oligonucleotide has the structure:

5′ T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′ (SEQ ID NO: 316) or 5′T*C*G*C*G*A*C_G*T*T*C*G*C*G*C_G*C*G*C*G 3′. (SEQ ID NO: 317)

In another aspect an oligonucleotide having the formula5′T*T*GX₁X₂TGX₃X₄T*T*T*T*N₁₀T*T*T*T*T*T*T3′ (SEQ ID NO:318) is provided.N₁₀ is between 4 and 8 nucleotides in length and includes at least 1 C_Gmotif. X₁, X₂, X₃ and, X₄ are independently C or G. The oligonucleotidehas a length of 24-40 nucleotides.

In some embodiments N₁₀ includes at least 2 or 3 CG motifs. In otherembodiments the oligonucleotide has one of the following structures:

5′ T*T*G*C_G*T*G*C_G*T*T*T*T*G*A*C_G*T*T*T*T*T*T*T 3′ (SEQ ID NO: 319)or 5′ T*T*G*G_C*T*G*G_C*T*T*T*T*G*A*C_G*T*T*T*T*T*T*T 3′.(SEQ ID NO: 320)

In other embodiments, the oligonucleotide has the structure:

5′ T*C*G*C_G*A*C*G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′. (SEQ ID NO: 321)

In some aspects the ODN is an oligonucleotide having a sequence selectedfrom the group consisting of

CGTCGTTTTGACGTTTTGTCGTTT, (SEQ ID NO: 333) GTCGTTTTGACGTTTTGTCGTT,(SEQ ID NO: 334) TCGTTTTGACGTTTTGTCGTT, (SEQ ID NO: 335)CGTTTTGACGTTTTGTCGTT, (SEQ ID NO: 336) GTTTTGACGTTTTGTCGTT,(SEQ ID NO: 337) TTTTGACGTTTTGTCGTT, (SEQ ID NO: 338) TTTGACGTTTTGTCGTT,(SEQ ID NO: 339) TTGACGTTTTGTCGTT, (SEQ ID NO: 340) TGACGTTTTGTCGTT,(SEQ ID NO: 341) GACGTTTTGTCGTT, (SEQ ID NO: 342) ACGTTTTGTCGTT,(SEQ ID NO: 343) GTTTTGTCGTT, (SEQ ID NO: 344) GTTTTGTCGTT,(SEQ ID NO: 345) TTTTGTCGTT, (SEQ ID NO: 346) TTTGTCGTT, TTGTCGTT,(SEQ ID NO: 347) TCGTCGTTTTGACGTTTTGTCGT, TCGTCGTTTTGACGTTTTGTCG,(SEQ ID NO: 348) TCGTCGTTTTGACGTTTTGTC, (SEQ ID NO: 349)TCGTCGTTTTGACGTTTTGT, (SEQ ID NO: 350) TCGTCGTTTTGACGTTTTG,(SEQ ID NO: 351) TCGTCGTTTTGACGTTTT, (SEQ ID NO: 352) TCGTCGTTTTGACGTTT,(SEQ ID NO: 353) TCGTCGTTTTGACGTT, (SEQ ID NO: 354) TCGTCGTTTTGACGT,(SEQ ID NO: 355) TCGTCGTTTTGACG, (SEQ ID NO: 356) TCGTCGTTTTGAC,(SEQ ID NO: 357) TCGTCGTTTTGA, (SEQ ID NO: 358) TCGTCGTTTTG,(SEQ ID NO: 359) TCGTCGTTTT, (SEQ ID NO: 360) TCGTCGTTT, TCGTCGTT,(SEQ ID NO: 361) CGTCGTTTTGACGTTTTGTGGT, GTCGTTTTGACGTTTTTGTCG,(SEQ ID NO: 362) TCGTTTTGACGTTTTGTC, (SEQ ID NO: 363) CGTTTTGACGTTTTGT,(SEQ ID NO: 364) GTTTTGACGTTTTG, (SEQ ID NO: 365) TTTTGACGTTTT,(SEQ ID NO: 366) TTTGACGTTT, (SEQ ID NO: 367) and TTGACGTT.

In another aspect the invention is an oligonucleotide comprising anoctameric sequence comprising at least one YZ dinucleotide having aphosphodiester or phosphodiester-like internucleotide linkage, and atleast 4 T nucleotides, wherein Y is a pyrimidine or modified pyrimidine,wherein Z is a guanosine or modified guanosine, and wherein theoligonucleotide includes at least one stabilized internucleotidelinkage.

Y may be an unmethylated C. Z may be a guanosine. In some embodiments Yis cytosine or a modified cystosine bases selected from the groupconsisting of 5-methyl cytosine, 5-methyl-isocytosine,5-hydroxy-cytosine, 5-halogeno cytosine, uracil, N4-ethyl-cytosine,5-fluoro-uracil, and hydrogen. In other embodiments Z is guanine or amodified guanine base selected from the group consisting of7-deazaguanine, 7-deaza-7-substituted guanine (such as7-deaza-7-(C₂-C₆)alkynylguanine), 7-deaza-8-substituted guanine,hypoxanthine, 2,6-diaminopurine, 2-aminopurine, purine, 8-substitutedguanine such as 8-hydroxyguanine, and 6-thioguanine, 2-aminopurine, andhydrogen.

In some embodiments the octameric sequence includes a TTTT motif. Inother embodiments the octameric sequence includes two YZ dinucleotides.Optionally both YZ dinucleotides have a phosphodiester orphosphodiester-like internucleotide linkage.

In some embodiments the octameric sequence is selected from the groupconsisting of T*C-G*T*C-G*T*T, C-G*T*C-G*T*T*T, G*T*C-G*T*T*T*T,T*C-G*T*T*T*T*G, C-G*T*T*T*T*G*A, T*T*T*T*G*A*C-G, T*T*T*G*A*C-G*T,T*T*G*A*C-G*T*T, T*G*A*C-G*T*T*T, G*A*C-G*T*T*T*T, A*C-G*T*T*T*T*G,C-G*T*T*T*T*G*T, T*T*T*T*G*T*C-G, T*T*T*G*T*C-G*T, G*T*T*T*T*G*T*C, andT*T*G*T*C-G*T*T, wherein * refers to the presence of a stabilizedinternucleotide linkage, and wherein _ refers to the presence of aphosphodiester internucleotide linkage.

In other embodiments the oligonucleotide has a length of 8-40nucleotides.

The phosphodiester-like linkage may be boranophosphonate ordiastereomerically pure Rp phosphorothioate. Optionally the stabilizedinternucleotide linkages are phosphorothioate, phosphorodithioate,methylphosphonate, methylphosphorothioate, or any combination thereof.

The oligonucleotide may have a 3′-3′ linkage with one or two accessible5′ ends. In some preferred embodiments the oligonucleotide has twoaccessible 5′ ends, each of which are 5′TCG.

In another aspect of the invention an oligonucleotide comprising: 5′TCGTCGTTTTGACGTTTTGTCGTT 3′ (SEQ ID NO: 368) is provided. At least oneCG dinucleotide has a phosphodiester or phosphodiester-likeinternucleotide linkage, and the oligonucleotide includes at least onestabilized internucleotide linkage.

In other aspects the invention is an oligonucleotide comprising: 5′GNC3′, wherein N is a nucleic aid sequence of 4-10 nucleotides in lengthand is at least 50% T and does not include a CG dinucleotide, and theoligonucleotide includes at least one stabilized internucleotidelinkage. In one embodiment N includes a TTTT motif. In other embodimentsthe oligonucleotide is selected from the group consisting ofG*T*T*T*T*G*T*C and G*T*T*T*T*G*A*C, wherein * refers to the presence ofa stabilized internucleotide linkage.

In another aspect the invention provides an immunostimulatory nucleicacid molecule having at least one internal pyrimidine-purine (YZ)dinucleotide and optionally pyrimidine-guansosine (YG) dinucleotide anda chimeric backbone, wherein the at least one internal YZ dinucleotidehas a phosphodiester or phosphodiester-like internucleotide linkage,wherein optionally each additional internal YZ dinucleotide has aphosphodiester, phosphodiester-like, or stabilized internucleotidelinkage, and wherein all other internucleotide linkages are stabilized.In one embodiment the immunostimulatory nucleic acid comprises aplurality of internal YZ dinucleotides each having a phosphodiester orphosphodiester-like internucleotide linkage. In one embodiment everyinternal YZ dinucleotide has a phosphodiester or phosphodiester-likeinternucleotide linkage.

In one embodiment the immunostimulatory nucleic acid molecule isselected from the group consisting of:

*A*C_G*T*C_G*T*T*T*T*C_G*T*C_G*T*T, (SEQ ID NO: 1)G*C_G*T*C_G*A*C_G*T*C_G*A*C_G*C, (SEQ ID NO: 2)G*C_G*T*C_G*T*T*T*T*C_G*T*C_G*C, (SEQ ID NO: 3)T*C*C*A*T_G*A*C_G*T*T*C*C*T_G*A*T*G*C, (SEQ ID NO: 4)T*C*G*T*C*G*T*T*T*T*C*G*T*C_G*T*T, (SEQ ID NO: 5)T*C*G*T*C*G*T*T*T*T*C_G*G*C_G*G*C*C_G*C*C*G, (SEQ ID NO: 6)T*C*G*T*C*G*T*T*T*T*C_G*T*G_G*T*T, (SEQ ID NO: 7)T*C*G*T*C_G*T*T*T*C_G*T*C_G*T*T, (SEQ ID NO: 8)T*C*G*T*C_G*T*T*T*T*C*G*T*C*G*T*T, (SEQ ID NO: 9)T*C*G*T*C_G*T*T*T*T*C*G*T*C_G*T*T, (SEQ ID NO: 10)T*C*G*T*C_G*T*T*T*T*C_G*T*C*G*T*T, (SEQ ID NO: 11)T*C_7*T*C_7*T*T*T*T_G*T*C_G*T*T*T*T_G*T*C_G*T*T, (SEQ ID NO: 12)T*C_7*T*C_G*T*T*T*T_G*T*C_G*T*T*T*T_G*T*C_7*T*T, (SEQ ID NO: 13)T*C_G*C*C_G*T*T*T*T*C_G*G*C_G*G*C*C_G*C*C*G, (SEQ ID NO: 14)T*C_G*T*C*G*T*T*T*T*A*C*G*A*C*G*T*C*G*C*G, (SEQ ID NO: 15)T*C_G*T*C*G*T*T*T*T*A*C*G*A*C*G*T*C*G*T*G, (SEQ ID NO: 16)T*C_G*T*C*G*T*T*T*T*A*C*G*G*C*G*C*C*G*C*G*C*C*G, (SEQ ID NO: 17)T*C_G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G, (SEQ ID NO: 21)T*C_G*T*C*G*T*T*T*T*C*G*T*C*G*T*T, (SEQ ID NO: 22)T*C_G*T*C*G*T*T*T*T*C*G*T*C_G*T*T, (SEQ ID NO: 23)T*C_G*T*C*G*T*T*T*T*C_G*T*C*G*T*T, (SEQ ID NO: 24)T*C_G*T*C*G*T*T*T*T*G*C*G*A*C*G*T*C*G*C*G, (SEQ ID NO: 25)T*C_G*T*C*G*T*T*T*T*T*C*G*A*C*G*T*C*G*A*G, (SEQ ID NO: 26)T*C_G*T*C*G*T*T*T*T*T*C*G*A*C*G*T*C*G*C*G, (SEQ ID NO: 27)T*C_G*T*C_7*T*T*T*T_G*T*C_G*T*T*T*T_7*T*C_G*T*T, (SEQ ID NO: 28)T*C_G*T*C_G*T*T*T*C_G*A*C*G*T*T, (SEQ ID NO: 29)T*C_G*T*C_G*T*T*T*C_G*A*C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 30)T*C_G*T*C_G*T*T*T*C_G*T*C_G*A*C_G*T*C_G*T*T*T*C_G*T*C*G, (SEQ ID NO: 31)T*C_G*T*C_G*T*T*T*C_G*T*C_G*A*T, (SEQ ID NO: 32)T*C_G*T*C_G*T*T*T*C_G*T*C_G*A*T*T, (SEQ ID NO: 33)T*C_G*T*C_G*T*T*T*C_G*T*C_G*T, (SEQ ID NO: 34)T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T, (SEQ ID NO: 35)T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T, (SEQ ID NO: 36T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 37)T*C_G*T*C_G*T*T*T*G*T*C*G*T*C*G*G*C*G*G*C*C*G*C*C*G, (SEQ ID NO: 38)T*C_G*T*C_G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G, (SEQ ID NO: 39)T*C_G*T*C_G*T*T*T*T*C*G*G*C*G*G*C*C*G*C*C*G, (SEQ ID NO: 40)T*C_G*T*C_G*T*T*T*T*C*G*T*C*G*T*T, (SEQ ID NO: 41)T*C_G*T*C_G*T*T*T*T*C_G*G*C_G*C_G*C_G*C*C*G, (SEQ ID NO: 42)  (SEQ ID NO: 43) T*C_G*T*C_G*T*T*T*T*C_G*T*C_G*T, (SEQ ID NO: 44)T*C_G*T*C_G*T*T*T*T*C_G*T*C_G*T*T, (SEQ ID NO: 45)T*C_G*T*C_G*T*T*T*T*C_G*T*T_G*T*T, (SEQ ID NO: 46)T*C_G*T*C_G*T*T*T*T*G*T*C_G*T*C_G*T*T*T*T, (SEQ ID NO: 47)T*C_G*T*C_G*T*T*T*T*T*T*T*T*C_G*T*C_G*T*T*T*T, (SEQ ID NO: 48)T*C_G*T*C_G*T*T*T*T*T_G*T*C_G*T*T, (SEQ ID NO: 49)T*C_G*T*C_G*T*T*T*T*T_G*T*T_G*T*T, (SEQ ID NO: 50)T*C_G*T*C_G*T*T*T*T_7*T*C_7*T*T*T*T_G*T*C_G*T*T, (SEQ ID NO: 51)T*C_G*T*C_G*T*T*T*T_G*A*C_G*T*T, (SEQ ID NO: 52)T*C_G*T*C_G*T*T*T*T_G*A*C_G*T*T*T*T, (SEQ ID NO: 53)T*C_G*T*C_G*T*T*T*T_G*A*C_G*T*T*T*T*G*T*C*G*T*T, (SEQ ID NO: 54)T*C_G*T*C_G*T*T*T*T_G*A*C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 55)T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T, (SEQ ID NO: 56)T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 241)T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T*T*T_7*T*C_7*T*T, (SEQ ID NO: 58)T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T*T*T_G*T*C_G*T*T, (SEQ ID NO: 59)T*C_G*T*C_G*T*T*T*U G*T*C_G*T*T*T, (SEQ ID NO: 60)T*C_G*T*C_G*T*T*T*U_G*T*C_G*T*T*T*T_G*T*C_G*T*T, (SEQ ID NO: 61)T*C_G*T*C_G*T*T*T_G*G_G*T*C_G*T, (SEQ ID NO: 62)T*C_G*T*C_G*T*T*T_G*C_G*T*C_G*T*T, (SEQ ID NO: 63)T*C_G*T*C_G*T*T*T_G*T*C_G*T, (SEQ ID NO: 64)T*C_G*T*C_G*T*T*T_G*T*C_G*T*T, (SEQ ID NO: 65)T*C_G*T*C_G*U*U*U*C_G*T*C_G*U*U*U*U_G*T*C_G*T*T, (SEQ ID NO: 66)T*C_G*T*T*T*T*G*T*C_G*T*T*T*T, (SEQ ID NO: 67)T*C_G*T*T*T*T*G*T*C_G*T*T*T*T*T*T*T*T, (SEQ ID NO: 68)T*C_G*T*T*T*T*T*T*T*T*C_G*T*T*T*T, (SEQ ID NO: 69)T*C_G*T*T_G*T*T*T*T*C_G*T*C_G*T*T, (SEQ ID NO: 70)T*C_G*T*T_G*T*T*T*T*C_G*T*T_G*T*T, (SEQ ID NO: 71)T*C_G*T*T_G*T*T*T*T*T_G*T*C_G*T*T, (SEQ ID NO: 72)T*C_G*T*T_G*T*T*T*T*T_G*T*T_G*T*T, (SEQ ID NO: 73)T*C_G*U*C_G*T*T*T*T_G*T*C_G*T*T*T*U_G*U*C_G*T*T, (SEQ ID NO: 74)T*G*T*C_G*T*T*G*T*C_G*T*T*G*T*C_G*T*T*G*T*C_G*T*T, (SEQ ID NO: 75)T*G*T*C_G*T*T*G*T*C_G*T*T_G*T*C_G*T*T_G*T*C_G*T*T, (SEQ ID NO: 76)T*G*T*C_G*T*T*T*C_G*T*C_G *T*T, (SEQ ID NO: 77)T*G*T*C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 78)T*T*A*G*T*T*C_G*T*A*G*T*T*C*T*T*C_G*T*T, (SEQ ID NO: 79)T*T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T, (SEQ ID NO: 80)T*T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T*T, (SEQ ID NO: 81)T*T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T, (SEQ ID NO: 82)T*T*C_G*T*T*C*T*T*A*G*T*T*C_G*T*A*G*T*T, (SEQ ID NO: 83)T*T*T*C_G*A*C_G*T*C_G*T*T*T, (SEQ ID NO: 84)T*T*T*T*C_G*T*C_G*T*T*T*T*G*T*C_G*T*C_G*T, (SEQ ID NO: 85)T*T*T*T*C_G*T*C_G*T*T*T*T*G*T*C_G*T*C_G*T*T*T*T, (SEQ ID NO: 86)T*T*T*T*C_G*T*C_G*T*T*T*T*T*T*T*T*C_G*T*C_G*T, (SEQ ID NO: 87)T*T*T*T*C_G*T*C_G*T*T*T*T*T*T*T*T*C_G*T*C_G*T*T*T*T, (SEQ ID NO: 88)T*T*T*T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*C_G*T*T*T*T, (SEQ ID NO: 89)T*T*T*T*C_G*T*T*T*T*G*T*C_G*T, (SEQ ID NO: 90)T*T*T*T*C_G*T*T*T*T*G*T*C_G*T*T*T*T, (SEQ ID NO: 91)T*T*T*T*C_G*T*T*T*T*T*T*T*T*C_G*T, (SEQ ID NO: 92)T*T*T*T*C_G*T*T*T*T*T*T*T*T*C_G*T*T*T*T, (SEQ ID NO: 93)T*T*T*T*C_G_T*T*T*T_G*T*C_G*T*T*T*T, (SEQ ID NO: 94)T*T*T*T*T*T*T*T*C_G*T*T*T*T*G*T*C_G*T, (SEQ ID NO: 95)T*T_G*T*C_G*T*T*T*T*C_G*T*C_G*T*T, (SEQ ID NO: 96)T*T_G*T*C_G*T*T*T*T*C_G*T*T_G*T*T, (SEQ ID NO: 97)T*T_G*T*C_G*T*T*T*T*T_G*T*C_G*T*T, (SEQ ID NO: 98) andT*T_G*T*C_G*T*T*T*T*T_G*T*T_G*T* T, (SEQ ID NO: 99)wherein * represents phosphorothioate, _ represents phosphodiester, Urepresents 2′-deoxyuracil, and 7 represents 7-deazaguanine.

In one embodiment the immunostimulatory nucleic acid molecule isselected from the group consisting of:

T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 100)T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T, (SEQ ID NO: 101)T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T, (SEQ ID NO: 102)T*G*T*C_G*T*T*G*T*C_G*T*T_G*T*C_G*T*T_G*T*C_G*T*T (SEQ ID NO: 103) andT*C_G*T*C_G*T*T*T*T*C*G*G*C*G*G*C*C*G*C*C*G, (SEQ ID NO: 104)wherein * represents phosphorothioate and _ represents phosphodiester.

In another aspect the invention provides an immunostimulatory nucleicacid molecule comprising a chimeric backbone and at least one sequenceN₁YGN₂, wherein independently for each sequence N₁YGN₂ YG is an internalpyrimidine-guanosine (YG) dinucleotide, N₁ and N₂ are each, independentof the other, any nucleotide, and wherein for the at least one sequenceN₁YGN₂ and optionally for each additional sequence N₁YGN₂: the YGdinucleotide has a phosphodiester or phosphodiester-like internucleotidelinkage, and N₁ and Y are linked by a phosphodiester orphosphodiester-like internucleotide linkage when N₁ is an internalnucleotide, G and N₂ are linked by a phosphodiester orphosphodiester-like internucleotide linkage when N₂ is an internalnucleotide, or N₁ and Y are linked by a phosphodiester orphosphodiester-like internucleotide linkage when N₁ is an internalnucleotide and G and N₂ are linked by a phosphodiester orphosphodiester-like internucleotide linkage when N₂ is an internalnucleotide, wherein all other internucleotide linkages are stabilized.

In one embodiment the immunostimulatory nucleic acid comprises aplurality of the sequence N₁YGN₂, wherein for each sequence N₁YGN₂: theYG dinucleotide has a phosphodiester or phosphodiester-likeinternucleotide linkage, and N₁ and Y are linked by a phosphodiester orphosphodiester-like internucleotide linkage when N₁ is an internalnucleotide, G and N₂ are linked by a phosphodiester orphosphodiester-like internucleotide linkage when N₂ is an internalnucleotide, or N₁ and Y are linked by a phosphodiester orphosphodiester-like internucleotide linkage when N₁ is an internalnucleotide and G and N₂ are linked by a phosphodiester orphosphodiester-like internucleotide linkage when N₂ is an internalnucleotide.

In one embodiment the immunostimulatory nucleic acid molecule isselected from the group consisting of:

T*C_G*T*C_G*T*T*T*T*G*T*C_G*T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 105)T*C_G*T*C_G*T*T*T*T*G*T*C_G*T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 106)T*C_G*T*C_G*T*T*T*T*G*T*C_G*T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 107)T*C_G*T*C_G*T*T*T*T*G*T*C_G_T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 108)T*C_G*T*C_G*T*T*T*T*G*T*C_G_T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 109)T*C_G*T*C_G*T*T*T*T*G*T*C_G_T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 110)T*C_G*T*C_G*T*T*T*T*G*T*C_G_T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 111)T*C_G*T*C_G*T*T*T*T*G*T_C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 112)T*C_G*T*C_G*T*T*T*T*G*T_C_G*T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 113)T*C_G*T*C_G*T*T*T*T*G*T_C_G*T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 114)T*C_G*T*C_G*T*T*T*T*G*T_C_G*T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 115)T*C_G*T*C_G*T*T*T*T*G*T_C_G_T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 116)T*C_G*T*C_G*T*T*T*T*G*T_C_G_T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 117)T*C_G*T*C_G*T*T*T*T*G*T_C_G_T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 118)T*C_G*T*C_G*T*T*T*T*G*T_C_G_T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 119)T*C_G*T*C_G_T*T*T*T*G*T*C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 120)T*C_G*T*C_G_T*T*T*T*G*T*C_G*T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 121)T*C_G*T*C_G_T*T*T*T*G*T*C_G*T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 122)T*C_G*T*C_G_T*T*T*T*G*T*C_G*T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 123)T*C_G*T*C_G_T*T*T*T*G*T*C_G_T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 124)T*C_G*T*C_G_T*T*T*T*G*T*C_G_T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 125)T*C_G*T*C_G_T*T*T*T*G*T*C_G_T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 126)T*C_G*T*C_G_T*T*T*T*G*T*C_G_T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 127)T*C_G*T*C_G_T*T*T*T*G*T_C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 128)T*C_G*T*C_G_T*T*T*T*G*T_C_G*T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 129)T*C_G*T*C_G_T*T*T*T*G*T_C_G*T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 130)T*C_G*T*C_G_T*T*T*T*G*T_C_G*T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 131)T*C_G*T*C_G_T*T*T*T*G*T_C_G_T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 132)T*C_G*T*C_G_T*T*T*T*G*T_C_G_T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 133)T*C_G*T*C_G_T*T*T*T*G*T_C_G_T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 134)T*C_G*T*C_G_T*T*T*T*G*T_C_G_T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 135)T*C_G*T_C_G*T*T*T*T*G*T*C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 136)T*C_G*T_C_G*T*T*T*T*G*T*C_G*T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 137)T*C_G*T_C_G*T*T*T*T*G*T*C_G*T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 138)T*C_G*T_C_G*T*T*T*T*G*T*C_G*T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 139)T*C_G*T_C_G*T*T*T*T*G*T*C_G_T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 140)T*C_G*T_C_G*T*T*T*T*G*T*C_G_T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 141)T*C_G*T_C_G*T*T*T*T*G*T*C_G_T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 142)T*C_G*T_C_G*T*T*T*T*G*T*C_G_T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 143)T*C_G*T_C_G*T*T*T*T*G*T_C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 144)T*C_G*T_C_G*T*T*T*T*G*T_C_G*T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 145)T*C_G*T_C_G*T*T*T*T*G*T_C_G*T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 146)T*C_G*T_C_G*T*T*T*T*G*T_C_G*T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 147)T*C_G*T_C_G*T*T*T*T*G*T_C_G_T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 148)T*C_G*T_C_G*T*T*T*T*G*T_C_G_T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 149)T*C_G*T_C_G*T*T*T*T*G*T_C_G_T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 150)T*C_G*T_C_G*T*T*T*T*G*T_C_G_T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 151)T*C_G*T_C_G_T*T*T*T*G*T*C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 152)T*C_G*T_C_G_T*T*T*T*G*T*C_G*T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 153)T*C_G*T_C_G_T*T*T*T*G*T*C_G*T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 154)T*C_G*T_C_G_T*T*T*T*G*T*C_G*T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 155)T*C_G*T_C_G_T*T*T*T*G*T*C_G_T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 156)T*C_G*T_C_G_T*T*T*T*G*T*C_G_T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 157)T*C_G*T_C_G_T*T*T*T*G*T*C_G_T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 158)T*C_G*T_C_G_T*T*T*T*G*T*C_G_T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 159)T*C_G*T_C_G_T*T*T*T*G*T_C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 160)T*C_G*T_C_G_T*T*T*T*G*T_C_G*T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 161)T*C_G*T_C_G_T*T*T*T*G*T_C_G*T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 162)T*C_G*T_C_G_T*T*T*T*G*T_C_G*T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 163)T*C_G*T_C_G_T*T*T*T*G*T_C_G_T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 164)T*C_G*T_C_C_T*T*T*T*G*T_C_C_T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 165)T*C_G*T_C_G_T*T*T*T*G*T_C_G_T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 166)T*C_G*T_C_G_T*T*T*T*G*T_C_G_T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 167)T*C_G_T*C_G*T*T*T*T*G*T*C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 168)T*C_G_T*C_G*T*T*T*T*G*T*C_G*T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 169)T*C_G_T*C_G*T*T*T*T*G*T*C_G*T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 170)T*C_C_T*C_G*T*T*T*T*G*T*C_G*T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 171)T*C_G_T*C_G*T*T*T*T*G*T*C_C_T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 172)T*C_G_T*C_G*T*T*T*T*G*T*C_C_T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 173)T*C_G_T*C_G*T*T*T*T*C*T*C_G_T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 174)T*C_C_T*C_C*T*T*T*T*C*T*C_G_T*T*T*T*C*T_C_G_T*T, (SEQ ID NO: 175)T*C_C_T*C_G*T*T*T*T*G*T_C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 176)T*C_G_T*C_G*T*T*T*T*C*T_C_G*T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 177)T*C_G_T*C_G*T*T*T*T*C*T_C_G*T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 178)T*C_G_T*C_G*T*T*T*T*G*T_C_G*T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 179)T*C_G_T*C_G*T*T*T*T*G*T_C_G_T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 180)T*C_G_T*C_G*T*T*T*T*G*T_C_G_T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 181)T*C_G_T*C_G*T*T*T*T*G*T_C_G_T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 182)T*C_G_T*C_G*T*T*T*T*G*T_C_G_T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 183)T*C_G_T*C_G_T*T*T*T*G*T*C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 184)T*C_G_T*C_G_T*T*T*T*G*T*C_G*T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 185)T*C_G_T*C_G_T*T*T*T*G*T*C_G*T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 186)T*C_G_T*C_G_T*T*T*T*G*T*C_G*T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 187)T*C_G_T*C_G_T*T*T*T*G*T*C_G_T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 188)T*C_G_T*C_G_T*T*T*T*G*T*C_G_T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 189)T*C_G_T*C_G_T*T*T*T*G*T*C_G_T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 190)T*C_G_T*C_G_T*T*T*T*G*T*C_G_T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 191)T*C_G_T*C_G_T*T*T*T*G*T_C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 192)T*C_G_T*C_G_T*T*T*T*G*T_C_G*T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 193)T*C_G_T*C_G_T*T*T*T*G*T_C_G*T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 194)T*C_G_T*C_G_T*T*T*T*G*T_C_G*T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 195)T*C_G_T*C_G_T*T*T*T*G*T_C_G_T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 196)T*C_G_T*C_G_T*T*T*T*G*T_C_G_T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 197)T*C_G_T*C_G_T*T*T*T*G*T_C_G_T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 198)T*C_G_T*C_G_T*T*T*T*G*T_C_G_T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 199)T*C_G_T_C_G*T*T*T*T*G*T*C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 200)T*C_C_T_C_G*T*T*T*T*G*T*C_G*T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 201)T*C_C_T_C_G*T*T*T*T*G*T*C_G*T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 202)T*C_C_T_C_G*T*T*T*T*G*T*C_G*T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 203)T*C_G_T_C_G*T*T*T*T*G*T*C_G_T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 204)T*C_G_T_C_G*T*T*T*T*G*T*C_C_T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 205)T*C_G_T_C_G*T*T*T*T*G*T*C_G_T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 206)T*C_G_T_C_G*T*T*T*T*G*T*C_G_T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 207)T*C_G_T_C_G*T*T*T*T*G*T_C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 208)T*C_C_T_C_G*T*T*T*T*G*T_C_G*T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 209)T*C_G_T_C_G*T*T*T*T*G*T_C_G*T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 210)T*C_C_T_C_G*T*T*T*T*G*T_C_G*T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 211)T*C_G_T_C_G*T*T*T*T*G*T_C_G_T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 212)T*C_G_T_C_G*T*T*T*T*G*T_C_G_T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 213)T*C_G_T_C_G*T*T*T*T*G*T_C_G_T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 214)T*C_G_T_C_G*T*T*T*T*G*T_C_G_T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 215)T*C_G_T_C_G_T*T*T*T*G*T*C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 216)T*C_G_T_C_G_T*T*T*T*G*T*C_G*T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 217)T*C_G_T_C_G_T*T*T*T*G*T*C_G*T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 218)T*C_G_T_C_G_T*T*T*T*G*T*C_G*T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 219)T*C_G_T_C_G_T*T*T*T*G*T*C_G_T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 220)T*C_G_T_C_G_T*T*T*T*G*T*C_G_T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 221)T*C_G_T_C_G_T*T*T*T*G*T*C_G_T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 222)T*C_G_T_C_G_T*T*T*T*G*T*C_G_T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 223)T*C_G_T_C_G_T*T*T*T*G*T_C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 224)T*C_G_T_C_G_T*T*T*T*G*T_C_G*T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 225)T*C_G_T_C_G_T*T*T*T*G*T_C_G*T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 226)T*C_G_T_C_G_T*T*T*T*G*T_C_G*T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 227)T*C_G_T_C_G_T*T*T*T*G*T_C_G_T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 228)T*C_G_T_C_G_T*T*T*T*G*T_C_G_T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 229)T*C_G_T_C_G_T*T*T*T*G*T_C_G_T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 230) andT*C_G_T_C_G_T*T*T*T*G*T_C_G_T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 231)

-   -   wherein * represents phosphorothioate and _ represents        phosphodiester.

In one embodiment the immunostimulatory nucleic acid molecule isselected from the group consisting of:

T*C_G_T*C_G_T*T*T*T*G*T*C_G_T*T*T*T*G*T*C_G_T*T, (SEQ ID NO: 232)T*C_G*T_C_G*T*T*T*T*G*T_C_G*T*T*T*T*G*T_C_G*T*T, (SEQ ID NO: 233) andT*C_G_T_C_G_T*T*T*T*G*T_C_G_T*T*T*T*G*T_C_G_T*T, (SEQ ID NO: 234)wherein * represents phosphorothioate and _ represents phosphodiester.

In one embodiment the immunostimulatory nucleic acid molecule isselected from the group consisting of:

T*C*G*T*C*G*T*T*T_T_G*T*C*G*T*T*T_T_G*T*C*G*T*T, (SEQ ID NO: 235)T*C*G*T*C*G*T*T*T*T_G_T*C*G*T*T*T*T_G_T*C*G*T*T, (SEQ ID NO: 236) andT*C*G*T*C*G*T*T*T_T_G_T*C*G*T*T*T_T_C_T*C*G*T*T, (SEQ ID NO: 237)wherein * represents phosphorothioate and _ represents phosphodiester.

In one embodiment the immunostimulatory nucleic acid molecule isselected from the group consisting of:

T*C_G*T_C_G*T*T*T_T_G*T_C_G*T*T*T_T_G*T_C_G*T*T, (SEQ ID NO: 238)T*C_G_T*C_G_T*T*T*T_G_T*C_G_T*T*T*T_G_T*C_G_T*T, (SEQ ID NO: 239) andT*C_C_T_C_G_T*T*T_T_C_T_C_G_T*T*T_T_C_T_C_C_T*T, (SEQ ID NO: 240)wherein * represents phosphorothioate and _ represents phosphodiester.

In one embodiment the at least one internal YG dinucleotide having aphosphodiester or phosphodiester-like internucleotide linkage is CG. Inone embodiment the at least one internal YG dinucleotide having aphosphodiester or phosphodiester-like internucleotide linkage is TG.

In one embodiment the phosphodiester or phosphodiester-likeinternucleotide linkage is phosphodiester. In one embodiment thephosphodiester-like linkage is boranophosphonate or diastereomericallypure Rp phosphorothioate.

In one embodiment the stabilized internucleotide linkages are selectedfrom the group consisting of: phosphorothioate, phosphorodithioate,methylphosphonate, methylphosphorothioate, and any combination thereof.In one embodiment the stabilized internucleotide linkages arephosphorothioate.

In one embodiment the immunostimulatory nucleic acid molecule is aB-Class immunostimulatory nucleic acid molecule. In one embodiment theimmunostimulatory nucleic acid molecule is a C-Class immunostimulatorynucleic acid molecule.

In one embodiment the immunostimulatory nucleic acid molecule is 4-100nucleotides long. In one embodiment the immunostimulatory nucleic acidmolecule is 6-40 nucleotides long. In one embodiment theimmunostimulatory nucleic acid molecule is 6-19 nucleotides long.

In one embodiment the immunostimulatory nucleic acid molecule is not anantisense oligonucleotide, triple-helix-forming oligonucleotide, orribozyme.

In another aspect the invention provides an oligonucleotide whichcomprises

-   -   N₁-C_G-N₂-C_G-N₃        wherein N₁ and N₃ are each independently a nucleic acid sequence        1-20 nucleotides in length, wherein _ indicates an internal        phosphodiester or phosphodiester-like internucleotide linkage,        wherein N₂ is independently a nucleic acid sequence 0-20        nucleotides in length, and wherein G-N₂-C includes 1 or 2        stabilized linkages.

In another aspect the invention provides an oligonucleotide whichcomprises

-   -   N₁-C_G-N₂-C_G-N₃        wherein N₁ and N₃ are each independently a nucleic acid sequence        1-20 nucleotides in length, wherein _ indicates an internal        phosphodiester or phosphodiester-like internucleotide linkage,        wherein N₂ is independently a nucleic acid sequence 4-20        nucleotides in length, and        wherein G-N₂-C includes at least 5 stabilized linkages.

In another aspect the invention provides an oligonucleotide whichcomprises

-   -   N₁-C_G-N₂-C_G-N₃        wherein N₁, N₂, and N₃ are each independently a nucleic acid        sequence of 0-20 nucleotides in length and wherein _ indicates        an internal phosphodiester or phosphodiester-like        internucleotide linkage, wherein the oligonucleotide is not an        antisense oligonucleotide, triple-helix-forming oligonucleotide,        or ribozyme.

In another aspect the invention provides a an oligonucleotide whichcomprises

-   -   X₁-N₁-(GTCGTT)_(n)-N₂-X₂ (SEQ ID NOS:18-20 and 57)        wherein N₁ and N₂ are each independently a nucleic acid sequence        of 0-20 nucleotides in length, wherein n=2 or n=4-6, wherein X₁        and X₂ are each independently a nucleic acid sequence having        phosphorothioate internucleotide linkages of 3-10 nucleotides,        wherein N₁-(GTCGTT)_(n)-N₂ includes at least one phosphodiester        internucleotide linkage, and wherein 3′ and 5′ nucleotides of        the oligonucleotide do not include a poly-G, poly-A, poly-T, or        poly-C sequence.

In one embodiment the nucleic acid has a backbone comprising deoxyriboseor ribose.

In one embodiment the oligonucleotide has a backbone comprisingdeoxyribose or ribose.

In one embodiment the oligonucleotide is in a pharmaceutical compositionoptionally comprising a pharmaceutically acceptable carrier.

In one embodiment the oligonucleotide further comprises an adjuvant or acytokine.

In one embodiment the oligonucleotide further comprises an antigen,wherein the oligonucleotide is a vaccine adjuvant.

In one embodiment the antigen is selected from the group consisting of:a viral antigen, a bacterial antigen, a fungal antigen, a parasiticantigen, and a tumor antigen. In one embodiment the antigen is encodedby a nucleic acid vector. In one embodiment the antigen is a peptideantigen. In one embodiment the antigen is covalently linked to theoligonucleotide or immunostimulatory nucleic acid molecule. In anotherembodiment the antigen is not covalently linked to the oligonucleotideor immunostimulatory nucleic acid molecule.

In another aspect the invention provides a method for identifying arelative potency or toxicity of a test immunostimulatory nucleic acidmolecule. The method involves selecting a reference immunostimulatorynucleic acid having a reference sequence, a stabilized backbone, and areference immunostimulatory potency or toxicity; selecting a testimmunostimulatory nucleic acid having the reference sequence, aphosphodiester or phosphodiester-like linkage in place of a stabilizedlinkage between Y and N of at least one internal YN dinucleotide in thereference sequence, wherein Y is a pyrimidine and N is any nucleotide,and having a test immunostimulatory potency or toxicity; and comparingthe test immunostimulatory potency or toxicity to the referenceimmunostimulatory potency or toxicity to identify the relative potencyor toxicity of a test immunostimulatory nucleic acid molecule.

In one embodiment the test immunostimulatory nucleic acid is a morepotent inducer of TLR9 signaling activity than the referenceimmunostimulatory nucleic acid.

In one embodiment the test immunostimulatory nucleic acid is a morepotent inducer of type 1 interferon than the reference immunostimulatorynucleic acid.

In one embodiment the test immunostimulatory nucleic acid is a morepotent inducer of IP-10 than the reference immunostimulatory nucleicacid.

In one embodiment YN is YG. In one embodiment the at least one internalYG dinucleotide is CG. In one embodiment the at least one internal YGdinucleotide is TG.

In one embodiment the test immunostimulatory nucleic acid comprises aplurality of internal YG dinucleotides each having a phosphodiester orphosphodiester-like internucleotide linkage. In one embodiment the atleast one internal YG dinucleotide is every internal YG dinucleotide.

In one embodiment the phosphodiester or phosphodiester-like linkage isphosphodiester. In one embodiment the phosphodiester-like linkage isboranophosphonate or diastereomerically pure Rp phosphorothioate.

In one embodiment the stabilized backbone comprises a plurality ofinternucleotide linkages selected from the group consisting of:phosphorothioate, phosphorodithioate, methylphosphonate,methylphosphorothioate, and any combination thereof. In one embodimentthe stabilized backbone comprises a plurality of phosphorothioateinternucleotide linkages.

In one embodiment the reference immunostimulatory nucleic acid moleculeis a B-Class immunostimulatory nucleic acid molecule. In one embodimentthe reference immunostimulatory nucleic acid molecule is a C-Classimmunostimulatory nucleic acid molecule.

In one embodiment the reference immunostimulatory nucleic acid moleculeis 4-100 nucleotides long. In one embodiment the referenceimmunostimulatory nucleic acid molecule is 6-40 nucleotides long. In oneembodiment the reference immunostimulatory nucleic acid molecule is 6-19nucleotides long.

In another aspect the invention provides a method for designing astabilized immunostimulatory nucleic acid molecule less than 20nucleotides long. The method involves selecting a sequence 6-19nucleotides long, wherein the sequence includes at least one internal CGdinucleotide; selecting a phosphodiester or phosphodiester-like linkagebetween C and G of at least one internal CG dinucleotide; independentlyselecting a phosphodiester, phosphodiester-like, or stabilized linkagebetween C and G of each additional internal CG dinucleotide; andselecting a stabilized linkage for all other internucleotide linkages.

In another aspect, the invention is a method for treating or preventingallergy or asthma. The method is performed by administering to a subjectan immunostimulatory CpG oligonucleotide described herein in aneffective amount to treat or prevent allergy or asthma. In oneembodiment the oligonucleotide is administered to a mucosal surface. Inother embodiments the oligonucleotide is administered in an aerosolformulation. Optionally the oligonucleotide is administeredintranasally.

A method for inducing cytokine production is provided according toanother aspect of the invention. The method is performed byadministering to a subject an immunostimulatory CpG oligonucleotidedescribed herein in an effective amount to induce a cytokine selectedfrom the group consisting of IL-6, IL-8, IL-12, IL-18, TNF, IFN-α,chemokines, and IFN-γ.

In another aspect the invention is a composition of the CpGimmunostimulatory oligonucleotides described herein in combination withan antigen or other therapeutic compound, such as an anti-microbialagent. The anti-microbial agent may be, for instance, an anti-viralagent, an anti-parasitic agent, an anti-bacterial agent or ananti-fungal agent.

A composition of a sustained release device including the CpGimmunostimulatory oligonucleotides described herein is providedaccording to another aspect of the invention.

The composition may optionally include a pharmaceutical carrier and/orbe formulated in a delivery device. In some embodiments the deliverydevice is selected from the group consisting of cationic lipids, cellpermeating proteins, and sustained release devices. In one embodimentthe sustained release device is a biodegradable polymer or amicroparticle.

According to another aspect of the invention a method of stimulating animmune response is provided. The method involves administering a CpGimmunostimulatory oligonucleotide to a subject in an amount effective toinduce an immune response in the subject. Preferably the CpGimmunostimulatory oligonucleotide is administered orally, locally, in asustained release device, mucosally, systemically, parenterally, orintramuscularly. When the CpG immunostimulatory oligonucleotide isadministered to the mucosal surface it may be delivered in an amounteffective for inducing a mucosal immune response or a systemic immuneresponse. In preferred embodiments the mucosal surface is selected fromthe group consisting of an oral, nasal, rectal, vaginal, and ocularsurface.

In some embodiments the method includes exposing the subject to anantigen wherein the immune response is an antigen-specific immuneresponse. In some embodiments the antigen is selected from the groupconsisting of a tumor antigen, a viral antigen, a bacterial antigen, aparasitic antigen and a peptide antigen.

CpG immunostimulatory oligonucleotides are capable of provoking a broadspectrum of immune response. For instance these CpG immunostimulatoryoligonucleotides can be used to redirect a Th2 to a Th1 immune response.CpG immunostimulatory oligonucleotides may also be used to activate animmune cell, such as a lymphocyte (e.g., B and T cells), a dendriticcell, and an NK cell. The activation can be performed in vivo, in vitro,or ex vivo, i.e., by isolating an immune cell from the subject,contacting the immune cell with an effective amount to activate theimmune cell of the CpG immunostimulatory oligonucleotide andre-administering the activated immune cell to the subject. In someembodiments the dendritic cell presents a cancer antigen. The dendriticcell can be exposed to the cancer antigen ex vivo.

The immune response produced by CpG immunostimulatory oligonucleotidesmay also result in induction of cytokine production, e.g., production ofIL-6, IL-8, IL-12, IL-18, TNF, IFN-α, chemokines, and IFN-γ.

In still another embodiment, the CpG immunostimulatory oligonucleotidesare useful for treating cancer. The CpG immunostimulatoryoligonucleotides are also useful according to other aspects of theinvention in preventing cancer (e.g., reducing a risk of developingcancer) in a subject at risk of developing a cancer. The cancer may beselected from the group consisting of biliary tract cancer, breastcancer, cervical cancer, choriocarcinoma, colon cancer, endometrialcancer, gastric cancer, intraepithelial neoplasms, lymphomas, livercancer, lung cancer (e.g. small cell and non-small cell), melanoma,neuroblastomas, oral cancer, ovarian cancer, pancreatic cancer, prostatecancer, rectal cancer, sarcomas, thyroid cancer, and renal cancer, aswell as other carcinomas and sarcomas. In some important embodiments,the cancer is selected from the group consisting of bone cancer, brainand CNS cancer, connective tissue cancer, esophageal cancer, eye cancer,Hodgkin's lymphoma, larynx cancer, oral cavity cancer, skin cancer, andtesticular cancer.

CpG immunostimulatory oligonucleotides may also be used for increasingthe responsiveness of a cancer cell to a cancer therapy (e.g., ananti-cancer therapy), optionally when the CpG immunostimulatoryoligonucleotide is administered in conjunction with an anti-cancertherapy. The anti-cancer therapy may be a chemotherapy, a vaccine (e.g.,an in vitro primed dendritic cell vaccine or a cancer antigen vaccine)or an antibody based therapy. This latter therapy may also involveadministering an antibody specific for a cell surface antigen of, forexample, a cancer cell, wherein the immune response results in antibodydependent cellular cytotoxicity (ADCC). In one embodiment, the antibodymay be selected from the group consisting of Ributaxin, Herceptin,Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMART M195, ATRAGEN,Ovarex, Bexxar, LDP-03, ior t6, MDX-210, MDX-11, MDX-22, OV103, 3622W94,anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE,Pretarget, NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD-72000, LymphoCide,CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2,MDX-260, ANA Ab, SMART 1D10Ab, SMART ABL 364 Ab and ImmuRAIT-CEA.

Thus, according to some aspects of the invention, a subject havingcancer or at risk of having a cancer is administered a CpGimmunostimulatory oligonucleotide and an anti-cancer therapy. In someembodiments, the anti-cancer therapy is selected from the groupconsisting of a chemotherapeutic agent, an immunotherapeutic agent and acancer vaccine.

In still another embodiment of the methods directed to preventing ortreating cancer, the subject may be further administered interferon-α.

The invention in other aspects relates to methods for preventing diseasein a subject. The method involves administering to the subject a CpGimmunostimulatory oligonucleotide on a regular basis to promote immunesystem responsiveness to prevent disease in the subject. Examples ofdiseases or conditions sought to be prevented using the prophylacticmethods of the invention include microbial infections (e.g., sexuallytransmitted diseases) and anaphylactic shock from food allergies.

In other aspects, the invention is a method for inducing an innateimmune response by administering to the subject a CpG immunostimulatoryoligonucleotide in an amount effective for activating an innate immuneresponse.

According to another aspect of the invention a method for treating orpreventing a viral or retroviral infection is provided. The methodinvolves administering to a subject having or at risk of having a viralor retroviral infection, an effective amount for treating or preventingthe viral or retroviral infection of any of the compositions of theinvention. In some embodiments the virus is caused by a hepatitis viruse.g., hepatitis B, hepatitis C, HIV, herpes virus, or papillomavirus.

A method for treating or preventing a bacterial infection is providedaccording to another aspect of the invention. The method involvesadministering to a subject having or at risk of having a bacterialinfection, an effective amount for treating or preventing the bacterialinfection of any of the compositions of the invention. In one embodimentthe bacterial infection is due to an intracellular bacteria.

In another aspect the invention is a method for treating or preventing aparasite infection by administering to a subject having or at risk ofhaving a parasite infection, an effective amount for treating orpreventing the parasite infection of any of the compositions of theinvention. In one embodiment the parasite infection is due to anintracellular parasite. In another embodiment the parasite infection isdue to a non-helminthic parasite.

In some embodiments the subject is a human and in other embodiments thesubject is a non-human vertebrate selected from the group consisting ofa dog, cat, horse, cow, pig, turkey, goat, fish, monkey, chicken, rat,mouse, and sheep.

In another aspect the invention relates to a method for inducing a TH1immune response by administering to a subject any of the compositions ofthe invention in an effective amount to produce a TH1 immune response.

In another aspect the invention relates to a method for inducing animmune response, by administering to a subject in need thereof aneffective amount of an immunostimulatory oligonucleotide of5′T*C*G*T*X₁*T*T3′ wherein X₁ is 3-30 nucleotides, wherein * refers tothe presence of a stabilized internucleotide linkage, and wherein theoligonucleotide includes at least 2 phosphodiester internucleotidelinkages.

In another aspect, the invention relates to a method for treatingautoimmune disease by administering to a subject having or at risk ofhaving an autoimmune disease an effective amount for treating orpreventing the autoimmune disease of any of the compositions of theinvention.

In other embodiments the oligonucleotide is delivered to the subject inan effective amount to induce cytokine expression. Optionally thecytokine is selected from the group consisting of IL-6, TNFα, IFNα, IFNγand IP-10. In other embodiments the oligonucleotide is delivered to thesubject in an effective amount to shift the immune response to a Th1biased response form a Th2 biased response.

The invention is some aspects is a method for treating airwayremodeling, comprising: administering to a subject an oligonucleotidecomprising a CG dinucleotide, in an effective amount to treat airwayremodeling in the subject. In one embodiment the subject has asthma,chronic obstructive pulmonary disease, or is a smoker. In otherembodiments the subject is free of symptoms of asthma.

Use of an oligonucleotide of the invention for stimulating an immuneresponse is also provided as an aspect of the invention.

A method for manufacturing a medicament of an oligonucleotide of theinvention for stimulating an immune response is also provided.

In another aspect the invention relates to a method for stimulating animmune response, by administering to a subject an oligonucleotide of atleast 5 nucleotides in length in an effective amount to stimulate animmune response, wherein the oligonucleotide includes at least oneimmunostimulatory dinucleotide motif wherein the internucleotide linkagebetween the nucleotides of the dinucleotide has R chirality and whereinat least 70% of the other internucleotide linkages of theoligonucleotide have S chirality.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of graphs depicting levels of interferon-alpha (pg/ml)secreted from human PBMC following exposure of these cells to theoligonucleotides listed by number along the top X-axis of the graph anddepicted by a ▴ versus a positive control oligonucleotide depicted by a▪. The test oligonucleotides shown in FIG. 1A include SEQ ID NO: 322,SEQ ID NO: 323, and SEQ ID NO: 324 and the positive controloligonucleotide is SEQ ID NO: 242. The test oligonucleotides shown inFIG. 1B include SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, and SEQID NO: 328) and the positive control oligonucleotide is 5′ TCG TCG TTTTGA CGT TTT GTC GTT 3′) (SEQ ID NO: 329). The concentration ofoligonucleotide used to produce a particular data point is depictedalong the X-axis (μM). The data shown represents the mean of six donors.Below the graphs the level of Interferon-alpha (pg/ml) secreted by cellstreated with a negative control (medium) is listed for each experiment.

FIG. 2 is a set of graphs depicting levels of IL-10 (pg/ml) secretedfrom human PBMC following exposure of these cells to theoligonucleotides listed by number along the top X-axis of the graph anddepicted by a ▴ versus a positive control oligonucleotide depicted by a▪. The test oligonucleotides shown in FIG. 2A include (SEQ ID NO: 322),SEQ ID NO: 323, and SEQ ID NO: 324 and the positive controloligonucleotide is SEQ ID NO: 242. The test oligonucleotides shown inFIG. 2B include SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, and SEQID NO: 328 and the positive control oligonucleotide is SEQ ID NO: 329.The concentration of oligonucleotide used to produce a particular datapoint is depicted along the X-axis (μM). The data shown represents themean of six donors. Below the graphs the level of IL-10 (pg/ml) secretedby cells treated with a negative control (medium) is listed for eachexperiment.

FIG. 3 is a set of graphs depicting levels of TNF-alpha (pg/ml) secretedfrom human PBMC following exposure of these cells to theoligonucleotides listed by number along the top X-axis of the graph anddepicted by a ▴ versus a positive control oligonucleotide depicted by a▪. The test oligonucleotides shown in FIG. 3A include SEQ ID NO: 322,SEQ ID NO: 323, and SEQ ID NO: 324 and the positive controloligonucleotide is SEQ ID NO: 329. The test oligonucleotides shown inFIG. 3B include SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, and SEQID NO: 328 and the positive control oligonucleotide is SEQ ID NO: 329.The concentration of oligonucleotide used to produce a particular datapoint is depicted along the X-axis (μM). The data shown represents themean of three donors. Below the graphs the level of TNF-alpha (pg/ml)secreted by cells treated with a negative control (medium) and with LPSis listed for each experiment.

FIG. 4 is a set of graphs depicting levels of IL-6 (pg/ml) secreted fromhuman PBMC following exposure of these cells to the oligonucleotideslisted by number along the top X-axis of the graph and depicted by a ▴versus a positive control oligonucleotide depicted by a ▪. The testoligonucleotides shown in FIG. 4A include SEQ ID NO: 322, SEQ ID NO:323, and SEQ ID NO: 324 and the positive control oligonucleotide is SEQID NO: 329 (with a complete phosphorothioate modified backbone). Thetest oligonucleotides shown in FIG. 4B include SEQ ID NO: 325, SEQ IDNO: 326, SEQ ID NO: 327, and SEQ ID NO: 328 and the positive controloligonucleotide is SEQ ID NO: 329. The concentration of oligonucleotideused to produce a particular data point is depicted along the X-axis(μM). The data shown represents the mean of three donors. Below thegraphs the level of IL-6 (pg/ml) secreted by cells treated with anegative control (medium) and with LPS is listed for each experiment.

FIG. 5 is a set of graphs depicting levels of interferon-gamma (pg/ml)secreted from human PBMC following exposure of these cells to theoligonucleotides listed by number along the top X-axis of the graph anddepicted by a ▴ versus a positive control oligonucleotide depicted by a●. The test oligonucleotides shown in FIG. 5A include SEQ ID NO: 322,SEQ ID NO: 323, and SEQ ID NO: 324 and the positive controloligonucleotide is SEQ ID NO: 329. The test oligonucleotides shown inFIG. 5B include SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, and SEQID NO: 328 and the positive control oligonucleotide is SEQ ID NO: 329.The concentration of oligonucleotide used to produce a particular datapoint is depicted along the X-axis (μM). The data shown represents themean of three donors. Below the graphs the level of interferon-gamma(pg/ml) secreted by cells treated with a negative (medium) and with LPSis listed for each experiment.

FIG. 6 is a set of graphs depicting levels of CD69 expression (MFI) onNK cells as an indicator of NK cell activation following exposure ofthese cells to the oligonucleotides listed by number along the topX-axis of the graph and depicted by a ▴ versus a positive controloligonucleotide depicted by a ▪. The test oligonucleotides shown in FIG.6A include SEQ ID NO: 322, SEQ ID NO: 323, and SEQ ID NO: 324 and thepositive control oligonucleotide is SEQ ID NO: 329. The testoligonucleotides shown in FIG. 6B include SEQ ID NO: 325, SEQ ID NO:326, SEQ ID NO: 327, and SEQ ID NO: 328 and the positive controloligonucleotide is SEQ ID NO: 329. The concentration of oligonucleotideused to produce a particular data point is depicted along the X-axis(μM). The data shown represents the mean of three donors. Below thegraphs the level of CD69 expression on NK cells treated with a negativecontrol (medium) and with LPS is listed for each experiment.

FIG. 7 is a set of graphs depicting levels of interferon-alpha (IFN-α)(7A) and IL-10 (7B) produced by human PBMC following exposure of thesecells to the oligonucleotide SEQ ID NO: 313 and depicted by a ▪ versus apositive control oligonucleotide SEQ ID NO: 242 depicted by a ●. Theconcentration of oligonucleotide used to produce a particular data pointis depicted along the X-axis (μM).

FIG. 8 is a set of graphs depicting levels of interferon-alpha (IFN-α)(8A) and IL-10 (8B) produced by human PBMC following exposure of thesecells to the oligonucleotide SEQ ID NO: 314 and depicted by a ▪ versus apositive control oligonucleotide SEQ ID NO: 242 depicted by a ●. Thenegative control ODN is SEQ ID No 330: tccaggacttctctcaggtt. Theconcentration of oligonucleotide used to produce a particular data pointis depicted along the X-axis (μM).

FIG. 9 is a set of graphs depicting levels of interferon-alpha (IFN-α)(9A) and IL-10 (9B) produced by human PBMC following exposure of thesecells to the oligonucleotide SEQ ID NO: 319 and depicted by a ▪ versus apositive control oligonucleotide SEQ ID NO: 242 depicted by a ●. Theconcentration of oligonucleotide used to produce a particular data pointis depicted along the X-axis (μM).

FIG. 10 is a set of graphs depicting levels of interferon-alpha (IFN-α)(10A) and IL-10 (10B) produced by human PBMC following exposure of thesecells to the oligonucleotide SEQ ID NO: 316 and depicted by a ▪ versus apositive control oligonucleotide SEQ ID NO: 242 depicted by a ●. Theconcentration of oligonucleotide used to produce a particular data pointis depicted along the X-axis (μM).

FIG. 11 is a set of graphs depicting levels of interferon-alpha (IFN-α)(11A) and IL-10 (11B) produced by human PBMC following exposure of thesecells to the oligonucleotide SEQ ID NO: 317 and depicted by a ▪ versus apositive control oligonucleotide SEQ ID NO: 242 depicted by a ●. Theconcentration of oligonucleotide used to produce a particular data pointis depicted along the X-axis (μM).

FIG. 12 is a set of graphs depicting levels of interferon-alpha (IFN-α)(12A) and IL-10 (12B) produced by human PBMC following exposure of thesecells to the oligonucleotide SEQ ID NO: 320 and depicted by a ▪ versus apositive control oligonucleotide SEQ ID NO: 242 depicted by ●. Theconcentration of oligonucleotide used to produce a particular data pointis depicted along the X-axis (μM).

FIG. 13 is a set of graphs depicting levels of CD86 expression on Bcells (13A) and CD80 expression on monocytes (13B) following exposure ofthese cells to the oligonucleotide SEQ ID NO: 313 and depicted by a ▪versus a positive control oligonucleotide SEQ ID NO: 242 depicted by a●. The concentration of oligonucleotide used to produce a particulardata point is depicted along the X-axis (μM).

FIG. 14 is a set of graphs depicting levels of CD86 expression on Bcells (14A) and CD80 expression on monocytes (14B) following exposure ofthese cells to the oligonucleotide SEQ ID NO: 314 and depicted by a ▪versus a positive control oligonucleotide SEQ ID NO: 242 depicted by ●.The concentration of oligonucleotide used to produce a particular datapoint is depicted along the X-axis (μM).

FIG. 15 is a set of graphs depicting levels of CD86 expression on Bcells (15A) and CD80 expression on monocytes (15B) following exposure ofthese cells to the oligonucleotide SEQ ID NO: 319 and depicted by a ▪versus a positive control oligonucleotide SEQ ID NO: 242 depicted by ●.The concentration of oligonucleotide used to produce a particular datapoint is depicted along the X-axis (μM).

FIG. 16 is a set of graphs depicting CD86 expression on B cells (16A)and CD80 expression on monocytes (16B) following exposure of these cellsto the oligonucleotide SEQ ID NO: 316 and compared with a positivecontrol oligonucleotide SEQ ID NO: 242, and oligonucleotide 5′ TCC AGGACT TCT CTC AGG TT 3′) SEQ ID NO: 330. The concentration ofoligonucleotide used to produce a particular data point is depictedalong the X-axis (μM).

FIG. 17 is a set of graphs depicting levels of interferon-alpha (IFN-α)(17A) and IL-10 (17B) produced by human PBMC following exposure of thesecells to the oligonucleotide SEQ ID NO: 321 in comparison to controloligonucleotide SEQ ID NO: 242 and to oligonucleotide SEQ ID NO: 330.The concentration of oligonucleotide used to produce a particular datapoint is depicted along the X-axis (μM).

FIG. 18 is a set of graphs depicting CD86 expression on B cells (18A)and CD80 expression on monocytes (18B) following exposure of these cellsto the oligonucleotide SEQ ID NO: 321 and compared with a positivecontrol oligonucleotide SEQ ID NO: 242, and oligonucleotide SEQ ID NO:330. The concentration of oligonucleotide used to produce a particulardata point is depicted along the X-axis (μM).

FIG. 19 is a set of graphs depicting CD86 expression on B cells (19A)and CD80 expression on monocytes (19B) following exposure of these cellsto the oligonucleotide SEQ ID NO: 317 and compared with a positivecontrol oligonucleotide SEQ ID NO: 242, and oligonucleotide SEQ ID NO:330. The concentration of oligonucleotide used to produce a particulardata point is depicted along the X-axis (μM).

FIG. 20 is a set of graphs depicting CD86 expression on B cells (20A)and CD80 expression on monocytes (20B) following exposure of these cellsto the oligonucleotide SEQ ID NO: 320 and compared with a positivecontrol oligonucleotide SEQ ID NO: 242, and oligonucleotide SEQ ID NO:330. The concentration of oligonucleotide used to produce a particulardata point is depicted along the X-axis (μM).

FIG. 21 is a graphic representation of a portion of a nucleic acidmolecule, depicting structural features including bases (B), sugars, andbackbone with a phosphodiester linkage (shown circled) between 5′cytidine and 3′ guanosine and adjacent phosphorothioate linkages.

FIG. 22 is bar graph depicting relative tissue amounts ofphosphorothioate (SEQ ID NO: 242), soft (SEQ ID NO: 294), and semi-soft(SEQ ID NO: 241) oligonucleotides in kidney, spleen, and liver 48 hoursafter subcutaneous injection into mice. Oligonucleotides SEQ ID NO: 242and SEQ ID NO: 241 have identical base sequences and differ in theirbackbone composition.

FIG. 23 shows Stimulation of human immune cells in vitro by induction ofcytokines IL-6, IL-10, IFNα and IP-10 FIG. 24 shows Stimulation ofmurine splenocytes in vitro by increased efficacy and/or potency as aninducer of TLR9-associated cytokines IL-6, IL-10, IL-12p40, IFNα, TNFαand IP-10, without detectable secretion of IL-1, IL-2, IL-4, IL-5 orGM-CSF.

FIG. 25 shows induced expression of TLR9-associated genes (IL-6, TNFα,IFNα, IFNγ and IP-10) in the lung by an ODN of the invention (SEQ ID No.313).

FIG. 26 shows the effects of CpG ODN on antigen-induced lymph nodedevelopment in mice in vivo.

FIG. 27 demonstrates that CpG ODN suppress a Th2 response to antigensensitization.

FIG. 28 shows the effects on antigen-induced IgE production in mice invivo.

FIG. 29 demonstrates that antigen challenge caused an increase in thetotal number of leukocytes, predominantly eosinophils, in the airwaylumen.

FIG. 30 shows that antigen challenge caused an increase in the totalnumber of leukocytes, predominantly eosinophils, in the airway lumen andthat this was suppressed by an ODN of the invention (SEQ ID No. 313) ina dose-related manner.

FIGS. 31 and 32 show that antigen challenge caused airwayhyperreactivity and that this was suppressed by an ODN of the invention(SEQ ID No. 313) in a dose-related manner.

FIG. 33 shows ODN concentrations in rat plasma following IV & ITadministration at 5 mg/kg. The plasma data shows that SEQ ID No. 313 iscleared more rapidly from plasma compared to SEQ ID No. 329 followingboth IV & IT administration.

FIG. 34 shows ODN concentrations in rat lungs following IV & ITadministration at 5 mg/kg. Following IV administration at the same doselevel, lung concentrations of SEQ ID No. 313 are lower than SEQ ID No.329 concentrations. After IT administration the difference is lessmarked. Lung data for SEQ ID No. 329 is only available for up to 48 hrspost-dose.

FIG. 35 shows ODN concentrations in rat kidneys following IV & ITadministration at 5 mg/kg. The kidney data indicates that absolutelevels of SEQ ID No. 313 in the kidneys are lower than corresponding SEQID No. 329 concentrations following both IV and IT administration.

The renal exposure to SEQ ID No. 313 after IT administration inparticular, is markedly reduced compared to exposure to SEQ ID No. 329at the same dose level.

FIG. 36 shows ODN concentrations in rat kidneys following IVadministration at 5 mg/kg.

FIG. 37 shows ODN concentrations in rat kidneys following ITadministration at 5 mg/kg.

FIG. 38 shows concentrations of SEQ ID No. 313 and its 8-mermetabolite(s) in rat kidneys following IV administration of SEQ ID No.313 at 5 mg/kg.

FIG. 39 shows concentrations of SEQ ID No. 313 and its 8-mermetabolite(s) in rat kidneys following IT administration of SEQ ID No.313 at 5 mg/kg.

FIG. 40 is a graph depicting stimulation index of sets of Semi-soft ODNcompared with fully phosphorothioate ODN having the same sequence.

FIG. 41 is a set of bar graphs depicting cytokine induction A & B(IP-10), C (IFN), and D & E (TNF) in response to the administration ofsoft (SEQ ID NO 294), semi-soft (SEQ ID NO 241), and fullyphosphorothioate ODN (SEQ ID NO 242).

FIG. 42 is a set of graphs depicting antibody and cytotoxic T lymphocyteactivity in response to the administration of soft (SEQ ID NO 294),semi-soft (SEQ ID NO 241), and fully phosphorothioate ODN (SEQ ID NO242).

FIG. 43 is a set of graphs depicting antitumor therapy in mice usingsemi-soft (SEQ ID NO 241) or fully phosphorothioate ODN (SEQ ID NO 242).FIGS. 43A and B depict the results in a renal cell carcinoma model.FIGS. 43C and D depict the results in a murine neuroblastoma model.FIGS. 43E and F depict the results in a murine non-small cell lungcancer model.

DETAILED DESCRIPTION

Soft and semi-soft immunostimulatory nucleic acids are providedaccording to the invention. The immunostimulatory oligonucleotides ofthe invention described herein, in some embodiments have improvedproperties including similar or enhanced potency, reduced systemicexposure to the kidney, liver and spleen, and may have reducedreactogenicity at injection sites. Although applicant is not bound by amechanism, it is believed that these improved properties are associatedwith the strategic placement within the immunostimulatoryoligonucleotides of phosphodiester or phosphodiester-like“internucleotide linkages”. The term “internucleotide linkage” as usedherein refers to the covalent backbone linkage joining two adjacentnucleotides in a nucleic acid molecule. The covalent backbone linkagewill typically be a modified or unmodified phosphate linkage, but othermodifications are possible. Thus a linear oligonucleotide that is nnucleotides long has a total of n−1 internucleotide linkages. Thesecovalent backbone linkages can be modified or unmodified in theimmunostimulatory oligonucleotides according to the teachings of theinvention.

In particular, phosphodiester or phosphodiester-like internucleotidelinkages involve “internal dinucleotides”. An internal dinucleotide ingeneral shall mean any pair of adjacent nucleotides connected by aninternucleotide linkage, in which neither nucleotide in the pair ofnucleotides is a terminal nucleotide, i.e., neither nucleotide in thepair of nucleotides is a nucleotide defining the 5′ or 3′ end of theoligonucleotide. Thus a linear oligonucleotide that is n nucleotideslong has a total of n-1 dinucleotides and only n-3 internaldinucleotides. Each internucleotide linkage in an internal dinucleotideis an internal internucleotide linkage. Thus a linear oligonucleotidethat is n nucleotides long has a total of n−1 internucleotide linkagesand only n-3 internal internucleotide linkages. The strategically placedphosphodiester or phosphodiester-like internucleotide linkages,therefore, refer to phosphodiester or phosphodiester-likeinternucleotide linkages positioned between any pair of nucleotides inthe nucleic acid sequence. In some embodiments the phosphodiester orphosphodiester-like internucleotide linkages are not positioned betweeneither pair of nucleotides closest to the 5′ or 3′ end.

The invention is based at least in some aspects on the surprisingdiscovery that the soft and semi-soft nucleic acids described hereinhave at least the same or in many cases possess greaterimmunostimulatory activity, in many instances, than corresponding fullystabilized immunostimulatory oligonucleotides having the same nucleotidesequence. This was unexpected because it is widely believed thatphosphorothioate oligonucleotides are generally more immunostimulatorythan unstabilized oligonucleotides. The results were surprising becauseit was expected that if the “softening” bond was placed between thecritical immunostimulatory motif, i.e. CG that the nucleic acid mighthave reduced activity because the nucleic acid would easily be brokendown into non-CG containing fragments in vivo. Contrary to theexpectations many of these nucleic acids actually had equivalent orbetter activity in vitro and in vivo. It appears that the soft andsemi-soft oligonucleotides are at least as potent as, if not more potentthan, their fully stabilized counterparts; the net immunostimulatoryeffect of soft and semi-soft oligonucleotides represents a balancebetween activity and stability. At high concentrations, the balanceappears to favor activity, i.e., potency dominates. At lowconcentrations, this balance appears to favor stability, i.e., therelative instability associated with nuclease susceptibility dominates.

The invention in one aspect relates to soft oligonucleotides. A softoligonucleotide is an immunostimulatory oligonucleotide having apartially stabilized backbone, in which phosphodiester orphosphodiester-like internucleotide linkages occur only within andimmediately adjacent to at least one internal pyrimidine-purinedinucleotide (YZ). Preferably YZ is YG, a pyrimidine-guanosine (YG)dinucleotide. The at least one internal YZ dinucleotide itself has aphosphodiester or phosphodiester-like internucleotide linkage. Aphosphodiester or phosphodiester-like internucleotide linkage occurringimmediately adjacent to the at least one internal YZ dinucleotide can be5′, 3′, or both 5′ and 3′ to the at least one internal YZ dinucleotide.Preferably a phosphodiester or phosphodiester-like internucleotidelinkage occurring immediately adjacent to the at least one internal YZdinucleotide is itself an internal internucleotide linkage. Thus for asequence N₁ YZ N₂, wherein N₁ and N₂ are each, independent of the other,any single nucleotide, the YZ dinucleotide has a phosphodiester orphosphodiester-like internucleotide linkage, and in addition (a) N₁ andY are linked by a phosphodiester or phosphodiester-like internucleotidelinkage when N₁ is an internal nucleotide, (b) Z and N₂ are linked by aphosphodiester or phosphodiester-like internucleotide linkage when N₂ isan internal nucleotide, or (c) N₁ and Y are linked by a phosphodiesteror phosphodiester-like internucleotide linkage when N₁ is an internalnucleotide and Z and N₂ are linked by a phosphodiester orphosphodiester-like internucleotide linkage when N₂ is an internalnucleotide.

Nonlimiting examples of soft oligonucleotides include those described bySEQ ID NOs 105-231, SEQ ID NOs 232-234, SEQ ID Nos 235-237, and SEQ IDNOs 238-240.

Soft oligonucleotides according to the instant invention are believed tobe relatively susceptible to nuclease cleavage compared to completelystabilized oligonucleotides. Without meaning to be bound to a particulartheory or mechanism, it is believed that soft oligonucleotides of theinvention are cleavable to fragments with reduced or noimmunostimulatory activity relative to full-length softoligonucleotides. Incorporation of at least one nuclease-sensitiveinternucleotide linkage, particularly near the middle of theoligonucleotide, is believed to provide an “off switch” which alters thepharmacokinetics of the oligonucleotide so as to reduce the duration ofmaximal immunostimulatory activity of the oligonucleotide. This can beof particular value in tissues and in clinical applications in which itis desirable to avoid injury related to chronic local inflammation orimmunostimulation, e.g., the kidney.

The invention in another aspect relates to semi-soft oligonucleotides. Asemi-soft oligonucleotide is an immunostimulatory oligonucleotide havinga partially stabilized backbone, in which phosphodiester orphosphodiester-like internucleotide linkages occur only within at leastone internal pyrimidine-purine (YZ) dinucleotide. Semi-softoligonucleotides generally possess increased immunostimulatory potencyrelative to corresponding fully stabilized immunostimulatoryoligonucleotides. For example, the immunostimulatory potency ofsemi-soft SEQ ID NO: 241 is 2-5 times that of all-phosphorothioate SEQID NO: 242, where the two oligonucleotides share the same nucleotidesequence and differ only as to internal YZ internucleotide linkages asfollows, where * indicates phosphorothioate and _ indicatesphosphodiester:

T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T*T*T*G*T*C_G*T*T, (SEQ ID NO: 241)T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T, (SEQ ID NO: 242)SEQ ID NO: 241 incorporates internal phophodiester internucleotidelinkages involving both CG and TG (both YZ) dinucleotides. Due to thegreater potency of semi-soft oligonucleotides, semi-softoligonucleotides can be used at lower effective concentations and havelower effective doses than conventional fully stabilizedimmunostimulatory oligonucleotides in order to achieve a desiredbiological effect.

Whereas fully stabilized immunostimulatory oligonucleotides can exhibitdose-response maxima, semi-soft oligonucleotides of the instantinvention appear to have monotonically increasing dose-response curves(as assayed by TLR9 stimulation) extending into higher concentrationsbeyond the optimal concentration for corresponding fully stabilizedimmunostimulatory oligonucleotides. Thus it is believed that semi-softoligonuncleotides of the instant invention can induce greaterimmunostimulation than fully stabilized immunostimulatoryoligonucleotides.

It has been discovered according to the instant invention that theimmunostimulatory activity of weakly immunostimulatory fully stabilizedoligonucleotides can be increased by incorporation of at least oneinternal YZ dinucleotide with a phosphodiester or phosphodiester-likeinternucleotide linkage. Thus it is possible to start with a weaklyimmunostimulatory oligonucleotide, having a fully stabilized backbone,and to improve its immunostimulatory activity by substituting aphosphodiester or phosphodiester-like internucleotide linkage for astabilized internucleotide linkage of at least one internal YGdinucleotide. For example, SEQ ID NO: 243 was found to have moreimmunostimulatory activity than its fully stabilized counterpart SEQ IDNO: 244, where SEQ ID NO: 244 is a relatively weak immunostimulatoryoligonucleotide compared to SEQ ID NO: 242:

T*G*T*C_G*T*T*G*T*C_G*T*T_G*T*C_G*T*T_G*T*C_G*T*T, (SEQ ID NO: 243)T*G*T*C*G*T*T*G*T*C*G*T*T*G*T*C*G*T*T*G*T*C*G*T*T, (SEQ ID NO: 244)

Whereas fully stabilized immunostimulatory nucleic acids less than 20nucleotides long can have modest immunostimulatory activity comparedwith longer (e.g., 24 nucleotides long) fully stabilizedoligonucleotides, semi-soft oligonucleotides as short as 16 nucleotideslong have been discovered to have immunostimulatory activity at leastequal to immunostimulatory activity of fully stabilized oligonucleotidesover 20 nucleotides long. For example, SEQ ID NO: 245 and 5602 (both16-mers with partial sequence similarity to SEQ ID NO: 242) exhibitimmunositmultory activity comparable to that of SEQ ID NO: 242 (24-mer).

T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T, (SEQ ID NO: 245)5602 T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T, (SEQ ID NO: 56)T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T, (SEQ ID NO: 242)

In some instances where a 6-mer phosphorothioate oligonucleotideappeared to lack immunostimulatory activity, substitution of even onephosphodiester internal YZ internucleotide linkage for aphosphorothioate linkage was found to yield a corresponding 6-mer withimmunostimulatory activity.

It is also believed that the foregoing properties of semi-softoligonucleotides generally increase with increasing “dose” ofphosphodiester or phosphodiester-like internucleotide linkages involvinginternal YZ dinucleotides. Thus it is believed, for example, thatgenerally for a given oligonucleotide sequence with five internal YZdinucleotides, an oligonucleotide with five internal phosphodiester orphosphodiester-like YZ internucleotide linkages is moreimmunostimulatory than an oligonucleotide with four internalphosphodiester or phosphodiester-like YG internucleotide linkages, whichin turn is more immunostimulatory than an oligonucleotide with threeinternal phosphodiester or phosphodiester-like YZ internucleotidelinkages, which in turn is more immunostimulatory than anoligonucleotide with two internal phosphodiester or phosphodiester-likeYZ internucleotide linkages, which in turn is more immunostimulatorythan an oligonucleotide with one internal phosphodiester orphosphodiester-like YZ internucleotide linkage. Importantly, inclusionof even one internal phosphodiester or phosphodiester-like YZinternucleotide linkage is believed to be advantageous over no internalphosphodiester or phosphodiester-like YZ internucleotide linkage. Inaddition to the number of phosphodiester or phosphodiester-likeinternucleotide linkages, the position along the length of the nucleicacid can also affect potency.

Nonlimiting examples of semi-soft oligonucleotides include thosedescribed by SEQ ID NOs 1-99 and 241 and SEQ ID NOs 100-104.

The immunostimulatory oligonucleotides of the present invention aregenerally protected from rapid degradation in the serum. Theimmunostimulatory oligonucleotides of the present invention are alsogenerally protected from rapid degradation in most tissues, with theexception of particular tissues with specific or excessive nucleaseactivity that are capable of degrading the immunostimulatoryoligonucleotides. This results in the reduction of immunostimulatoryoligonucleotides in those particular tissues, the accumulation of whichcould otherwise lead to undesirable effects from long-term therapyutilizing degradation-resistant oligonucleotides. The oligonucleotidesof the instant invention will generally include, in addition to thephosphodiester or phosphodiester-like internucleotide linkages atpreferred internal positions, 5′ and 3′ ends that are resistant todegradation. Such degradation-resistant ends can involve any suitablemodification that results in an increased resistance against exonucleasedigestion over corresponding unmodified ends. For instance, the 5′ and3′ ends can be stabilized by the inclusion there of at least onephosphate modification of the backbone. In a preferred embodiment, theat least one phosphate modification of the backbone at each end isindependently a phosphorothioate, phosphorodithioate, methylphosphonate,or methylphosphorothioate internucleotide linkage. In anotherembodiment, the degradation-resistant end includes one or morenucleotide units connected by peptide or amide linkages at the 3′ end.Yet other stabilized ends, including but not limited to those describedfurther below, are meant to be encompassed by the invention.

As described above, the oligonucleotides of the instant inventioninclude phosphodiester or phosphodiester-like linkages within andoptionally adjacent to internal YG dinucleotides. Such YG dinucleotidesare frequently part of immunostimulatory motifs. It is not necessary,however, that an oligonucleotide contain phosphodiester orphosphodiester-like linkages within every immunostimulatory motif. As anexample, an oligonucleotide such as

T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T, (SEQ ID NO: 242)with four CpG dinucleotides could have phosphodiester linkages betweenthe C and G of the second, third, or fourth CpG dinucleotide, and anycombination thereof. Additional phosphodiester or phosphodiester-likelinkages may also be maintained for even more rapid renal digestion ofthese otherwise “stabilized oligonucleotides”. For example, SEQ ID NO:242 further contains two internal TG dinucleotides, either or both ofwhich, alone or in combination with any one or combination of internalCG dinucleotides, can have phosphodiester or phosphodiester-likeinternucleotide linkages.

A phosphodiester internucleotide linkage is the type of linkagecharacteristic of nucleic acids found in nature. As shown in FIG. 20,the phosphodiester internucleotide linkage includes a phosphorus atomflanked by two bridging oxygen atoms and bound also by two additionaloxygen atoms, one charged and the other uncharged. Phosphodiesterinternucleotide linkage is particularly preferred when it is importantto reduce the tissue half-life of the oligonucleotide.

A phosphodiester-like internucleotide linkage is a phosphorus-containingbridging group that is chemically and/or diastereomerically similar tophosphodiester. Measures of similarity to phosphodiester includesusceptibility to nuclease digestion and ability to activate RNAse H.Thus for example phosphodiester, but not phosphorothioate,oligonucleotides are susceptible to nuclease digestion, while bothphosphodiester and phosphorothioate oligonucleotides activate RNAse H.In a preferred embodiment the phosphodiester-like internucleotidelinkage is boranophosphate (or equivalently, boranophosphonate) linkage.U.S. Pat. Nos. 5,177,198; 5,859,231; 6,160,109; 6,207,819; Sergueev etal., (1998) J Am Chem Soc 120:9417-27. In another preferred embodimentthe phosphodiester-like internucleotide linkage is diasteromericallypure Rp phosphorothioate. It is believed that diasteromerically pure Rpphosphorothioate is more susceptible to nuclease digestion and is betterat activating RNAse H than mixed or diastereomerically pure Spphosphorothioate. Stereoisomers of CpG oligonucleotides are the subjectof co-pending U.S. patent application Ser. No. 09/361,575 filed Jul. 27,1999, and published PCT application PCT/US99/17100 (WO 00/06588). It isto be noted that for purposes of the instant invention, the term“phosphodiester-like internucleotide linkage” specifically excludesphosphorodithioate and methylphosphonate internucleotide linkages.

The immunostimulatory nucleic acid molecules of the instant inventionhave chimeric backbone. For purposes of the instant invention, achimeric backbone refers to a partially stabilized backbone, wherein atleast one internucleotide linkage is phosphodiester orphosphodiester-like, and wherein at least one other internucleotidelinkage is a stabilized internucleotide linkage, wherein the at leastone phosphodiester or phosphodiester-like linkage and the at least onestabilized linkage are different. Since boranophosphonate linkages havebeen reported to be stabilized relative to phosphodiester linkages, forpurposes of the chimeric nature of the backbone, boranophosphonatelinkages can be classified either as phosphodiester-like or asstabilized, depending on the context. For example, a chimeric backboneaccording to the instant invention could in one embodiment include atleast one phosphodiester (phosphodiester or phosphodiester-like) linkageand at least one boranophosphonate (stabilized) linkage. In anotherembodiment a chimeric backbone according to the instant invention couldinclude boranophosphonate (phosphodiester or phosphodiester-like) andphosphorothioate (stabilized) linkages. A “stabilized internucleotidelinkage” shall mean an internucleotide linkage that is relativelyresistant to in vivo degradation (e.g., via an exo- or endo-nuclease),compared to a phosphodiester internucleotide linkage. Preferredstabilized internucleotide linkages include, without limitation,phosphorothioate, phosphorodithioate, methylphosphonate, and methylphosphoroth ioate. Other stabilized internucleotide linkages include,without limitation: peptide, alkyl, dephospho, and others as describedabove.

Modified backbones such as phosphorothioates may be synthesized usingautomated techniques employing either phosphoramidate or H-phosphonatechemistries. Aryl- and alkyl-phosphonates can be made, e.g., asdescribed in U.S. Pat. No. 4,469,863; and alkylphosphotriesters (inwhich the charged oxygen moiety is alkylated as described in U.S. Pat.No. 5,023,243 and European Patent No. 092,574) can be prepared byautomated solid phase synthesis using commercially available reagents.Methods for making other DNA backbone modifications and substitutionshave been described. Uhlmann E et al. (1990) Chem Rev 90:544; GoodchildJ (1990) Bioconjugate Chem 1:165. Methods for preparing chimericoligonucleotides are also known. For instance patents issued to Uhlmannet al have described such techniques.

Mixed backbone modified ODN may be synthesized using a commerciallyavailable DNA synthesizer and standard phosphoramidite chemistry. (F. E.Eckstein, “Oligonucleotides and Analogues—A Practical Approach” IRLPress, Oxford, UK, 1991, and M. D. Matteucci and M. H. Caruthers,Tetrahedron Lett. 21, 719 (1980)) After coupling, PS linkages areintroduced by sulfurization using the Beaucage reagent (R. P. Iyer, W.Egan, J. B. Regan and S. L. Beaucage, J. Am. Chem. Soc. 112, 1253(1990)) (0.075 M in acetonitrile) or phenyl acetyl disulfide (PADS)followed by capping with acetic anhydride, 2,6-lutidine intetrahydrofurane (1:1:8; v:v:v) and N-methylimidazole (16% intetrahydrofurane). This capping step is performed after thesulfurization reaction to minimize formation of undesired phosphodiester(PO) linkages at positions where a phosphorothioate linkage should belocated. In the case of the introduction of a phosphodiester linkage,e.g. at a CpG dinucleotide, the intermediate phosphorous-III is oxidizedby treatment with a solution of iodine in water/pyridine. After cleavagefrom the solid support and final deprotection by treatment withconcentrated ammonia (15 hrs at 50° C.), the ODN are analyzed by HPLC ona Gen-Pak Fax column (Millipore-Waters) using a NaCl-gradient (e.g.buffer A: 10 mM NaH₂PO₄ in acetonitrile/water=1:4/v:v pH 6.8; buffer B:10 mM NaH₂PO₄, 1.5 M NaCl in acetonitrile/water=1:4/v:v; 5 to 60% B in30 minutes at 1 ml/min) or by capillary gel electrophoresis. The ODN canbe purified by HPLC or by FPLC on a Source High Performance column(Amersham Pharmacia). HPLC-homogeneous fractions are combined anddesalted via a C18 column or by ultrafiltration. The ODN was analyzed byMALDI-TOF mass spectrometry to confirm the calculated mass.

The nucleic acids of the invention can also include other modifications.These include nonionic DNA analogs, such as alkyl- and aryl-phosphates(in which the charged phosphonate oxygen is replaced by an alkyl or arylgroup), phosphodiester and alkylphosphotriesters, in which the chargedoxygen moiety is alkylated. Nucleic acids which contain diol, such astetraethyleneglycol or hexaethyleneglycol, at either or both terminihave also been shown to be substantially resistant to nucleasedegradation.

The size (i.e., the number of nucleotide residues along the length ofthe nucleic acid) of the immunostimulatory oligonucleotide may alsocontribute to the stimulatory activity of the oligonucleotide. Forfacilitating uptake into cells immunostimulatory oligonucleotidespreferably have a minimum length of 6 nucleotide residues. Nucleic acidsof any size greater than 6 nucleotides (even many kb long) are capableof inducing an immune response according to the invention if sufficientimmunostimulatory motifs are present, since larger nucleic acids aredegraded inside of cells. It is believed by the instant inventors thatsemi-soft oligonucleotides as short as 4 nucleotides can also beimmunostimulatory if they can be delivered to the interior of the cell.In certain preferred embodiments according to the instant invention, theimmunostimulatory oligonucleotides are between 4 and 100 nucleotideslong. In typical embodiments the immunostimulatory oligonucleotides arebetween 6 and 40 nucleotides long. In certain preferred embodimentsaccording to the instant invention, the immunostimulatoryoligonucleotides are between 6 and 19 nucleotides long.

The oligonucleotides of the present invention are nucleic acids thatcontain specific sequences found to elicit an immune response. Thesespecific sequences that elicit an immune response are referred to as“immunostimulatory motifs”, and the oligonucleotides that containimmunostimulatory motifs are referred to as “immunostimulatory nucleicacid molecules” and, equivalently, “immunostimulatory nucleic acids” or“immunostimulatory oligonucleotides”. The immunostimulatoryoligonucleotides of the invention thus include at least oneimmunostimulatory motif. In a preferred embodiment the immunostimulatorymotif is an “internal immunostimulatory motif”. The term “internalimmunostimulatory motif” refers to the position of the motif sequencewithin a longer nucleic acid sequence, which is longer in length thanthe motif sequence by at least one nucleotide linked to both the 5′ and3′ ends of the immunostimulatory motif sequence.

In some embodiments of the invention the immunostimulatoryoligonucleotides include immunostimulatory motifs which are “CpGdinucleotides”. A CpG dinucleotide can be methylated or unmethylated. Animmunostimulatory nucleic acid containing at least one unmethylated CpGdinucleotide is a nucleic acid molecule which contains an unmethylatedcytosine-guanine dinucleotide sequence (i.e., an unmethylated 5′cytidine followed by 3′ guanosine and linked by a phosphate bond) andwhich activates the immune system; such an immunostimulatory nucleicacid is a CpG nucleic acid. CpG nucleic acids have been described in anumber of issued patents, published patent applications, and otherpublications, including U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806;6,218,371; 6,239,116; and 6,339,068. An immunostimulatory nucleic acidcontaining at least one methylated CpG dinucleotide is a nucleic acidwhich contains a methylated cytosine-guanine dinucleotide sequence(i.e., a methylated 5′ cytidine followed by a 3′ guanosine and linked bya phosphate bond) and which activates the immune system. In otherembodiments the immunostimulatory oligonucleotides are free of CpGdinucleotides. These oligonucleotides which are free of CpGdinucleotides are referred to as non-CpG oligonucleotides, and they havenon-CpG immunostimulatory motifs. The invention, therefore, alsoencompasses nucleic acids with other types of immunostimulatory motifs,which can be methylated or unmethylated. The immunostimulatoryoligonucleotides of the invention, further, can include any combinationof methylated and unmethylated CpG and non-CpG immunostimulatory motifs.

As to CpG nucleic acids, it has recently been described that there aredifferent classes of CpG nucleic acids. One class is potent foractivating B cells but is relatively weak in inducing IFN-α and NK cellactivation; this class has been termed the B class. The B class CpGnucleic acids typically are fully stabilized and include an unmethylatedCpG dinucleotide within certain preferred base contexts. See, e.g., U.S.Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and6,339,068. Another class is potent for inducing IFN-α and NK cellactivation but is relatively weak at stimulating B cells; this class hasbeen termed the A class. The A class CpG nucleic acids typically havestabilized poly-G sequences at 5′ and 3′ ends and a palindromicphosphodiester CpG dinucleotide-containing sequence of at least 6nucleotides. See, for example, published patent applicationPCT/US00/26527 (WO 01/22990). Yet another class of CpG nucleic acidsactivates B cells and NK cells and induces IFN-α; this class has beentermed the C-class. The C-class CpG nucleic acids, as firstcharacterized, typically are fully stabilized, include a B class-typesequence and a GC-rich palindrome or near-palindrome. This class hasbeen described in co-pending U.S. provisional patent application60/313,273, filed Aug. 17, 2001 and U.S. Ser. No. 10/224,523 filed onAug. 19, 2002, the entire contents of which are incorporated herein byreference. Some non limiting examples of C-Class nucleic acids include:

SEQ ID NO Sequence 275 T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 369T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 370T*C_G*G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 371T*C_G*G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 372T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G 373T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 374T*C_G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 375T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*C*G 316T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G

Thus, the invention in one aspect involves the finding that specificsub-classes of CpG immunostimulatory oligonucleotides having chimericbackbones are highly effective in mediating immune stimulatory effects.These CpG nucleic acids are useful therapeutically and prophylacticallyfor stimulating the immune system to treat cancer, infectious diseases,allergy, asthma, autoimmune disease, and other disorders and to helpprotect against opportunistic infections following cancer chemotherapy.The strong yet balanced, cellular and humoral immune responses thatresult from CpG stimulation reflect the body's own natural defensesystem against invading pathogens and cancerous cell.

The invention involves, in one aspect, the discovery that a subset ofCpG immunostimulatory oligonucleotides have improved immune stimulatoryproperties and reduced renal inflammatory effect. In some instances,renal inflammation has been observed in subjects that have beenadministered a completely phosphorothioate oligonucleotide. It isbelieved that the chimeric nucleic acids described herein produce lessrenal inflammation than fully phosphorothioate oligonucleotides.Additionally these oligonucleotides are highly effective in stimulatingan immune response. Thus, the phosphodiester region of the molecule didnot reduce it's effectivity.

The preferred CpG immunostimulatory oligonucleotides fall within one ofthe following 6 general formulas:

5′ T*C*G*T*CGTTTTGAN₁CGN₂*T*T 3′, (SEQ ID NO: 296) 5′T*C*G*(T*/A*)TN₃CGTTTTN₄CGN₅*T*T 3′, (SEQ ID NO: 301) 5′T*C*G*T*C*GNNNCGNCGNNNC*G*N*C*G*T*T 3′, (SEQ ID NO: 307) 5′T*C_G(N₆C_G N₇)₂₋₃T*C_G*T*T 3′, (SEQ ID NO: 311-312) 5′T*T*GX₁X₂TGX₃X₄T*T*T*T*N₁₀T*T*T*T*T*T*T 3′ (SEQ ID NO: 331) and 5′T*CGCGN₈CGCGC*GN₉ 3′. (SEQ ID NO: 332)

In these formulas N is any nucleotide, N₁ is 0-6 nucleotides, N₂ is 0-7nucleotides, N₃ is 0-4 nucleotides, N₄ is 1-5 nucleotides, N₅ is 0-7nucleotides, N₆ and N₇ are independently between 1 and 5 nucleotides inlength, N₈ is between 4 and 10 nucleotides in length, N₉ is between 0and 3 nucleotides in length and wherein N₁₀ is between 4 and 8nucleotides in length. X₁, X₂, X₃ and, X₄ are independently C or G. Theformulas define subsets of the class of CpG oligonucleotides whichdemonstrated excellent immune stimulating properties and yet were moresensitive to degradation within the body than fully phosphorothioatecontaining oligonucleotides. In the formula 5′ refers to the free 5′ endof the oligonucleotide and 3′ refers to the free 3′ end of theoligonucleotide.

The symbol * used in the formulas refers to the presence of a stabilizedinternucleotide linkage. The internucleotide linkages not marked withan * may be stabilized or unstabilized, as long as the oligonucleotideincludes at least 2-3 phosphodiester internucleotide linkages. In someembodiments it is preferred that the oligonucleotides include 3-6phosphodiester linkages. In some cases the linkages between the CGmotifs are phosphodiester and in other cases they are phosphorothioateor other stabilized linkages.

Other formulas include 5′ TCGTCGTTTTGACGTTTTGTCGTT 3′ (SEQ ID NO: 368),wherein at least one CG dinucleotide has a phosphodiester orphosphodiester-like internucleotide linkage, and the oligonucleotideincludes at least one stabilized internucleotide linkage and 5′GNC 3′,wherein N is a nucleic aid sequence of 4-10 nucleotides in length and isat least 50% T and does not include a CG dinucleotide, and theoligonucleotide includes at least one stabilized internucleotidelinkage.

In some embodiment the oligonucleotide has one of the followingstructures:

5′ T*C*G*T*C*G*TTTTGAN₁C*G*N₂*T*T 3′, (SEQ ID NO: 296) 5′T*C*G*T*C*G*T*T_T_T_GAN₁C*G*N₂*T*T 3′, (SEQ ID NO: 296) 5′T*C*G*T*C*G*T*T*T*T*GA_N₁C*G*N₂*T*T 3′, (SEQ ID NO: 296) 5′T*C*G*(T*/A*)TN₃CGTTTTN₄C*G*N₅*T*T 3′, (SEQ ID NO: 301) 5′T*C*G*A*T*N₃C*G*TTTTN₄C_G_*N₅*T*T 3′, (SEQ ID NO: 302) 5′T*C*G*T*T*N₃C_G_TTTTN₄CGN₅*T*T 3′, (SEQ ID NO: 303) 5′T*C*G*T*C*G*N*N*N*C_G_N_C_G_N*N*N*C*G*N*C*G*T*T 3′, (SEQ ID NO: 307) 5′T*C*G*T*C*G*T*T*A*C_G_N_C_G_T*T*A*C*G*N*C*G*T*T 3′, (SEQ ID NO: 308) or5′ T*C*G*T*C*G*N*N*N*C_G_T_C_G_N*N*N*C*G*T*C*G*T*T 3′. (SEQ ID NO: 309)

The symbol _ in these structures refers to the presence of aphosphodiester internucleotide linkage.

Some preferred examples of the structures include the following:

5′ T*C*G*T*C*G*T*T*T*T*G*A_C_C_G_G_T*T*C*G*T*G*T*T 3′, (SEQ ID NO: 327)5′ T*C*G*T*C*G*T*T*T*T*G_A_C*G*T*T*T*T*G*T*C*G*T*T 3′, (SEQ ID NO: 328)5′ T*C*G*T*C*G*T*T_T_T_G*A*C*G*T*T*T*T 3′, (SEQ ID NO: 324) 5′T*C*G*T*C*G*T*T_T_T_G*A*C*G*T*T 3′, (SEQ ID NO: 325) 5′T*C*G*A*T*C*G*T*T*T*T_T_C_G*T*G*C*G*T*T*T*T*T 3′, (SEQ ID NO: 323) 5′T*C*G*T*T*T*T*G*A_C_G_T*T*T*T*G*T*C*G*T*T 3′, (SEQ ID NO: 326) 5′T*C*G*T*C*G*T*T*A*C_G_T_C_G_T*T*A*C*G*T*C*G*T*T 3′, (SEQ ID NO: 322) 5′T*C_G*T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C_G*T*T 3′, (SEQ ID NO: 313) 5′T*C_G*A*C_G*T*T*T*T*G*T*C_G*T*T*T*T*G*T*C_G*T*T 3′, (SEQ ID NO: 314) 5′T*T*G*C_G*T*G*C_G*T*T*T*T*G*A*C_G*T*T*T*T*T*T*T 3′, (SEQ ID NO: 319) 5′T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′, (SEQ ID NO: 316) 5′T*C*G*C*G*A*C_G*T*T*C*G*C*G*C_G*C*G*C*G 3′, (SEQ ID NO: 317) 5′T*T*G*G_C*T*G*G_C*T*T*T*T*G*A*C_G*T*T*T*T*T*T*T 3′, (SEQ ID NO: 320) 5′T*C*G*C_G*A*C*G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3′, (SEQ ID NO: 321)T*C-G*T*C-G*T*T, C-G*T*C-G*T*T*T, G*T*C-G*T*T*T*T, T*C-G*T*T*T*T*G, C-G*T*T*T*T*G*A, T*T*T*T*G*A*C-G, T*T*T*G*A*C-G*T, T*T*G*A*C-G*T*T,T*G*A*C-G*T*T*T, G*A*C-G*T*T*T*T, A*C-G*T*T*T*T*G, C-G*T*T*T*T*G*T,T*T*T*T*G*T*C-G, T*T*T*G*T*C-G*T, G*T*T*T*T*G*T*C, or T*T*G*T*C-G*T*T.

The immunostimulatory oligonucleotides generally have a length in therange of between 4 and 100 and in some embodiments 10 and 40. The lengthmay be in the range of between 16 and 24 nucleotides.

The terms “nucleic acid” and “oligonucleotide” also encompass nucleicacids or oligonucleotides with substitutions or modifications, such asin the bases and/or sugars. For example, they include nucleic acidshaving backbone sugars that are covalently attached to low molecularweight organic groups other than a hydroxyl group at the 2′ position andother than a phosphate group or hydroxy group at the 5′ position. Thusmodified nucleic acids may include a 2′-O-alkylated ribose group. Inaddition, modified nucleic acids may include sugars such as arabinose or2′-fluoroarabinose instead of ribose. Thus the nucleic acids may beheterogeneous in backbone composition thereby containing any possiblecombination of polymer units linked together such as peptide-nucleicacids (which have an amino acid backbone with nucleic acid bases).

Nucleic acids also include substituted purines and pyrimidines such asC-5 propyne pyrimidine and 7-deaza-7-substituted purine modified bases.Wagner R W et al. (1996) Nat Biotechnol 14:840-4. Purines andpyrimidines include but are not limited to adenine, cytosine, guanine,thymine, 5-methylcytosine, 5-hydroxycytosine, 5-fluorocytosine,2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,and other naturally and non-naturally occurring nucleobases, substitutedand unsubstituted aromatic moieties. Other such modifications are wellknown to those of skill in the art.

The immunostimulatory oligonucleotides of the instant invention canencompass various chemical modifications and substitutions, incomparison to natural RNA and DNA, involving a phosphodiesterinternucleotide bridge, a β-D-ribose unit and/or a natural nucleotidebase (adenine, guanine, cytosine, thymine, uracil). Examples of chemicalmodifications are known to the skilled person and are described, forexample, in Uhlmann E et al. (1990) Chem Rev 90:543; “Protocols forOligonucleotides and Analogs” Synthesis and Properties & Synthesis andAnalytical Techniques, S. Agrawal, Ed, Humana Press, Totowa, USA 1993;Crooke S T et al. (1996) Annu Rev Pharmacol Toxicol 36:107-129; andHunziker J et al. (1995) Mod Synth Methods 7:331-417. An oligonucleotideaccording to the invention may have one or more modifications, whereineach modification is located at a particular phosphodiesterinternucleotide bridge and/or at a particular β-D-ribose unit and/or ata particular natural nucleotide base position in comparison to anoligonucleotide of the same sequence which is composed of natural DNA orRNA.

For example, the invention relates to an oligonucleotide which maycomprise one or more modifications and wherein each modification isindependently selected from:

-   a) the replacement of a phosphodiester internucleotide bridge    located at the 3′ and/or the 5′ end of a nucleotide by a modified    internucleotide bridge,-   b) the replacement of phosphodiester bridge located at the 3′ and/or    the 5′ end of a nucleotide by a dephospho bridge,-   c) the replacement of a sugar phosphate unit from the sugar    phosphate backbone by another unit,-   d) the replacement of a β-D-ribose unit by a modified sugar unit,    and-   e) the replacement of a natural nucleotide base by a modified    nucleotide base.

More detailed examples for the chemical modification of anoligonucleotide are as follows.

A phosphodiester internucleotide bridge located at the 3′ and/or the 5′end of a nucleotide can be replaced by a modified internucleotidebridge, wherein the modified internucleotide bridge is for exampleselected from phosphorothioate, phosphorodith ioate,NR¹R²-phosphoramidate, boranophosphate, α-hydroxybenzyl phosphonate,phosphate-(C₁-C₂₁)—O-alkyl ester,phosphate-[(C₆-C₂)aryl-(C₁-C₂₁)—O-alkyl]ester, (C₁-C₈)alkylphosphonateand/or (C₆-C₁₂)arylphosphonate bridges, (C₇-C₁₂)-α-hydroxymethyl-aryl(e.g., disclosed in WO 95/01363), wherein (C₆-C₁₂)aryl, (C₆-C₂₀)aryl and(C₆-C₁₄)aryl are optionally substituted by halogen, alkyl, alkoxy,nitro, cyano, and where R¹ and R² are, independently of each other,hydrogen, (C₁-C₁₈)-alkyl, (C₆-C₂₀)-aryl, (C₆-C₁₄)-aryl-(C₁-C₈)-alkyl,preferably hydrogen, (C₁-C₈)-alkyl, preferably (C₁-C₄)-alkyl and/ormethoxyethyl, or R¹ and R² form, together with the nitrogen atomcarrying them, a 5-6-membered heterocyclic ring which can additionallycontain a further heteroatom from the group O, S and N.

The replacement of a phosphodiester bridge located at the 3′ and/or the5′ end of a nucleotide by a dephospho bridge (dephospho bridges aredescribed, for example, in Uhlmann E and Peyman A in “Methods inMolecular Biology”, Vol. 20, “Protocols for Oligonucleotides andAnalogs”, S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16, pp.355 ff), wherein a dephospho bridge is for example selected from thedephospho bridges formacetal, 3′-thioformacetal, methylhydroxylamine,oxime, methylenedimethyl-hydrazo, dimethylenesulfone and/or silylgroups.

A sugar phosphate unit (i.e., a β-D-ribose and phosphodiesterinternucleotide bridge together forming a sugar phosphate unit) from thesugar phosphate backbone (i.e., a sugar phosphate backbone is composedof sugar phosphate units) can be replaced by another unit, wherein theother unit is for example suitable to build up a “morpholino-derivative”oligomer (as described, for example, in Stirchak E P et al. (1989)Nucleic Acids Res 17:6129-41), that is, e.g., the replacement by amorpholino-derivative unit; or to build up a polyamide nucleic acid(“PNA”; as described for example, in Nielsen P E et al. (1994) BioconjugChem 5:3-7), that is, e.g., the replacement by a PNA backbone unit,e.g., by 2-aminoethylglycine.

A β-ribose unit or a β-D-2′-deoxyribose unit can be replaced by amodified sugar unit, wherein the modified sugar unit is for exampleselected from β-D-ribose, α-D-2′-deoxyribose, L-2′-deoxyribose,2′-F-2′-deoxyribose, 2′-F-arabinose, 2′-O—(C₁-C₆)alkyl-ribose,preferably 2′-O—(C₁-C₆)alkyl-ribose is 2′-O-methylribose,2′-O—(C₂-C₆)alkenyl-ribose, 2′-[O—(C₁-C₆)alkyl-O—(C₁-C₆)alkyl]-ribose,2′-NH₂-2′-deoxyribose, β-D-xylo-furanose, α-arabinofuranose,2,4-dideoxy-β-D-erythro-hexo-pyranose, and carbocyclic (described, forexample, in Froehier J (1992) Am Chem Soc 114:8320) and/or open-chainsugar analogs (described, for example, in Vandendriessche et al. (1993)Tetrahedron 49:7223) and/or bicyclosugar analogs (described, forexample, in Tarkov M et al. (1993) Helv Chim Acta 76:481).

In some preferred embodiments the sugar is 2′-O-methylribose,particularly for one or both nucleotides linked by a phosphodiester orphosphodiester-like internucleotide linkage.

Nucleic acids also include substituted purines and pyrimidines such asC-5 propyne pyrimidine and 7-deaza-7-substituted purine modified bases.Wagner R W et al. (1996) Nat Biotechnol 14:840-4. Purines andpyrimidines include but are not limited to adenine, cytosine, guanine,and thymine, and other naturally and non-naturally occurringnucleobases, substituted and unsubstituted aromatic moieties.

A modified base is any base which is chemically distinct from thenaturally occurring bases typically found in DNA and RNA such as T, C,G, A, and U, but which share basic chemical structures with thesenaturally occurring bases. The modified nucleotide base may be, forexample, selected from hypoxanthine, uracil, dihydrouracil,pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil,5-(C₁-C₆)-alkyluracil, 5-(C₂-C₆)-alkenyluracil, 5-(C₂-C₆)-alkynyluracil,5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil,5-hydroxycytosine, 5-(C₁-C₆)-alkylcytosine, 5-(C₂-C₆)-alkenylcytosine,5-(C₂-C₆)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine,5-bromocytosine, N²-dimethylguanine, 2,4-diamino-purine, 8-azapurine, asubstituted 7-deazapurine, preferably 7-deaza-7-substituted and/or7-deaza-8-substituted purine, 5-hydroxymethylcytosine, N4-alkylcytosine,e.g., N4-ethylcytosine, 5-hydroxydeoxycytidine,5-hydroxymethyldeoxycytidine, N4-alkyldeoxycytidine, e.g.,N4-ethyldeoxycytidine, 6-thiodeoxyguanosine, and deoxyribonucleotides ofnitropyrrole, C5-propynylpyrimidine, and diaminopurine e.g.,2,6-diaminopurine, inosine, 5-methylcytosine, 2-aminopurine,2-amino-6-chloropurine, hypoxanthine or other modifications of a naturalnucleotide bases. This list is meant to be exemplary and is not to beinterpreted to be limiting.

In particular formulas described herein a set of modified bases isdefined. For instance the letter Y is used to refer to a nucleotidecontaining a cytosine or a modified cytosine. A modified cytosine asused herein is a naturally occurring or non-naturally occurringpyrimidine base analog of cytosine which can replace this base withoutimpairing the immunostimulatory activity of the oligonucleotide.Modified cytosines include but are not limited to 5-substitutedcytosines (e.g. 5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine,5-bromo-cytosine, 5-iodo-cytosine, 5-hydroxy-cytosine,5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine, and unsubstitutedor substituted 5-alkynyl-cytosine), 6-substituted cytosines,N4-substituted cytosines (e.g. N4-ethyl-cytosine), 5-aza-cytosine,2-mercapto-cytosine, isocytosine, pseudo-isocytosine, cytosine analogswith condensed ring systems (e.g. N,N′-propylene cytosine orphenoxazine), and uracil and its derivatives (e.g. 5-fluoro-uracil,5-bromo-uracil, 5-bromovinyl-uracil, 4-thio-uracil, 5-hydroxy-uracil,5-propynyl-uracil). Some of the preferred cytosines include5-methyl-cytosine, 5-fluoro-cytosine, 5-hydroxy-cytosine,5-hydroxymethyl-cytosine, and N4-ethyl-cytosine. In another embodimentof the invention, the cytosine base is substituted by a universal base(e.g. 3-nitropyrrole, P-base), an aromatic ring system (e.g.fluorobenzene or difluorobenzene) or a hydrogen atom (dSpacer).

The letter Z is used to refer to guanine or a modified guanine base. Amodified guanine as used herein is a naturally occurring ornon-naturally occurring purine base analog of guanine which can replacethis base without impairing the immunostimulatory activity of theoligonucleotide. Modified guanines include but are not limited to7-deazaguanine, 7-deaza-7-substituted guanine (such as7-deaza-7-(C₂-C₆)alkynylguanine), 7-deaza-8-substituted guanine,hypoxanthine, N2-substituted guanines (e.g. N2-methyl-guanine),5-amino-3-methyl-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione,2,6-diaminopurine, 2-aminopurine, purine, indole, adenine, substitutedadenines (e.g. N6-methyl-adenine, 8-oxo-adenine) 8-substituted guanine(e.g. 8-hydroxyguanine and 8-bromoguanine), and 6-thioguanine. Inanother embodiment of the invention, the guanine base is substituted bya universal base (e.g. 4-methyl-indole, 5-nitro-indole, and K-base), anaromatic ring system (e.g. benzimidazole or dichloro-benzimidazole,1-methyl-1H-[1,2,4]triazole-3-carboxylic acid amide) or a hydrogen atom(dSpacer).

The oligonucleotides may have one or more accessible 5′ ends. It ispossible to create modified oligonucleotides having two such 5′ ends.This may be achieved, for instance by attaching two oligonucleotidesthrough a 3′-3′ linkage to generate an oligonucleotide having one or twoaccessible 5′ ends. The 3′-3′-linkage may be a phosphodiester,phosphorothioate or any other modified internucleotide bridge. Methodsfor accomplishing such linkages are known in the art. For instance, suchlinkages have been described in Seliger, H.; et al., Oligonucleotideanalogs with terminal 3′-3′- and 5′-5′-internucleotidic linkages asantisense inhibitors of viral gene expression, Nucleotides & Nucleotides(1991), 10(1-3), 469-77 and Jiang, et al., Pseudo-cyclicoligonucleotides: in vitro and in vivo properties, Bioorganic &Medicinal Chemistry (1999), 7(12), 2727-2735.

Additionally, 3′3′-linked nucleic acids where the linkage between the3′-terminal nucleotides is not a phosphodiester, phosphorothioate orother modified bridge, can be prepared using an additional spacer, suchas tri- or tetra-ethylenglycol phosphate moiety (Durand, M. et al,Triple-helix formation by an oligonucleotide containing one (dA)12 andtwo (dT)12 sequences bridged by two hexaethylene glycol chains,Biochemistry (1992), 31(38), 9197-204, U.S. Pat. Nos. 5,658,738, and5,668,265). Alternatively, the non-nucleotidic linker may be derivedfrom ethanediol, propanediol, or from an abasic deoxyribose (dSpacer)unit (Fontanel, Marie Laurence et al., Sterical recognition by T4polynucleotide kinase of non-nucleosidic moieties 5′-attached tooligonucleotides; Nucleic Acids Research (1994), 22(11), 2022-7) usingstandard phosphoramidite chemistry. The non-nucleotidic linkers can beincorporated once or multiple times, or combined with each otherallowing for any desirable distance between the 3′-ends of the two ODNsto be linked.

It recently has been reported that CpG oligonucleotides appear to exerttheir immunostimulatory effect through interaction with Toll-likereceptor 9 (TLR9). Hemmi H et al. (2000) Nature 408:740-5. TLR9signaling activity thus can be measured in response to CpGoligonucleotide or other immunostimulatory nucleic acid by measuringNF-κB, NF-κB-related signals, and suitable events and intermediatesupstream of NF-κB.

For use in the instant invention, the oligonucleotides of the inventioncan be synthesized de novo using any of a number of procedures wellknown in the art. For example, the b-cyanoethyl phosphoramidite method(Beaucage, S. L., and Caruthers, M. H., Tet. Let. 22:1859, 1981);nucleotide H-phosphonate method (Garegg et al., Tet. Let. 27:4051-4054,1986; Froehler et al., Nucl. Acid. Res. 14:5399-5407, 1986,; Garegg etal., Tet. Let. 27:4055-4058, 1986, Gaffney et al., Tet. Let.29:2619-2622, 1988). These chemistries can be performed by a variety ofautomated nucleic acid synthesizers available in the market. Theseoligonucleotides are referred to as synthetic oligonucleotides. Anisolated oligonucleotide generally refers to an oligonucleotide which isseparated from components which it is normally associated with innature. As an example, an isolated oligonucleotide may be one which isseparated from a cell, from a nucleus, from mitochondria or fromchromatin.

The oligonucleotides are partially resistant to degradation (e.g., arestabilized). A “stabilized oligonucleotide molecule” shall mean anoligonucleotide that is relatively resistant to in vivo degradation(e.g. via an exo- or endo-nuclease). Nucleic acid stabilization can beaccomplished via backbone modifications. Oligonucleotides havingphosphorothioate linkages provide maximal activity and protect theoligonucleotide from degradation by intracellular exo- andendo-nucleases. Other modified oligonucleotides include phosphodiestermodified nucleic acids, combinations of phosphodiester andphosphorothioate nucleic acid, methylphosphonate,methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinationsthereof.

Modified backbones such as phosphorothioates may be synthesized usingautomated techniques employing either phosphoramidate or H-phosphonatechemistries. Aryl- and alkyl-phosphonates can be made, e.g., asdescribed in U.S. Pat. No. 4,469,863; and alkylphosphotriesters (inwhich the charged oxygen moiety is alkylated as described in U.S. Pat.No. 5,023,243 and European Patent No. 092,574) can be prepared byautomated solid phase synthesis using commercially available reagents.Methods for making other DNA backbone modifications and substitutionshave been described (e.g., Uhlmann, E. and Peyman, A., Chem. Rev.90:544, 1990; Goodchild, J., Bioconjugate Chem. 1:165, 1990).

Other stabilized oligonucleotides include: nonionic DNA analogs, such asalkyl- and aryl-phosphates (in which the charged phosphonate oxygen isreplaced by an alkyl or aryl group), phosphodiester andalkylphosphotriesters, in which the charged oxygen moiety is alkylated.Nucleic acids which contain diol, such as tetraethyleneglycol orhexaethyleneglycol, at either or both termini have also been shown to besubstantially resistant to nuclease degradation.

While CpG effects in mice are well characterized, information regardingthe human system is limited. CpG phosphorothioate oligonucleotides withstrong stimulatory activity in the mouse system show lower activity onhuman and other non-rodent immune cells. In the examples the developmentof a potent human CpG motif and the characterization of its effects andmechanisms of action on human PBMC, e.g., B-cells, and NK-cells isdescribed. DNA containing these CpG motifs and partially modifiedbackbones strongly stimulated human peripheral blood cells to produceIL-6, IL-10, IP-10, TNF-α, IFN-α, and IFN-γ. IFN-γ was increased overcontrol levels. NK cells and T cells were also induced to expressincreased levels of CD69.

It has been discovered according to the invention that the subsets ofCpG immunostimulatory oligonucleotides have dramatic immune stimulatoryeffects on human cells such as NK cells, suggesting that these CpGimmunostimulatory oligonucleotides are effective therapeutic agents forhuman vaccination, cancer immunotherapy, asthma immunotherapy, generalenhancement of immune function, enhancement of hematopoietic recoveryfollowing radiation or chemotherapy, autoimmune disease and other immunemodulatory applications.

Thus the CpG immunostimulatory oligonucleotides are useful in someaspects of the invention as a vaccine for the treatment of a subject atrisk of developing allergy or asthma, an infection with an infectiousorganism or a cancer in which a specific cancer antigen has beenidentified. The CpG immunostimulatory oligonucleotides can also be givenwithout the antigen or allergen for protection against infection,allergy or cancer, and in this case repeated doses may allow longer termprotection. A subject at risk as used herein is a subject who has anyrisk of exposure to an infection causing pathogen or a cancer or anallergen or a risk of developing cancer. For instance, a subject at riskmay be a subject who is planning to travel to an area where a particulartype of infectious agent is found or it may be a subject who throughlifestyle or medical procedures is exposed to bodily fluids which maycontain infectious organisms or directly to the organism or even anysubject living in an area where an infectious organism or an allergenhas been identified. Subjects at risk of developing infection alsoinclude general populations to which a medical agency recommendsvaccination with a particular infectious organism antigen. If theantigen is an allergen and the subject develops allergic responses tothat particular antigen and the subject may be exposed to the antigen,i.e., during pollen season, then that subject is at risk of exposure tothe antigen. A subject at risk of developing allergy or asthma includesthose subjects that have been identified as having an allergy or asthmabut that don't have the active disease during the CpG immunostimulatoryoligonucleotide treatment as well as subjects that are considered to beat risk of developing these diseases because of genetic or environmentalfactors.

A subject at risk of developing a cancer is one who has a highprobability of developing cancer. These subjects include, for instance,subjects having a genetic abnormality, the presence of which has beendemonstrated to have a correlative relation to a higher likelihood ofdeveloping a cancer and subjects exposed to cancer causing agents suchas tobacco, asbestos, or other chemical toxins, or a subject who haspreviously been treated for cancer and is in apparent remission. When asubject at risk of developing a cancer is treated with an antigenspecific for the type of cancer to which the subject is at risk ofdeveloping and a CpG immunostimulatory oligonucleotide, the subject maybe able to kill the cancer cells as they develop. If a tumor begins toform in the subject, the subject will develop a specific immune responseagainst the tumor antigen.

In addition to the use of the CpG immunostimulatory oligonucleotides forprophylactic treatment, the invention also encompasses the use of theCpG immunostimulatory oligonucleotides for the treatment of a subjecthaving an infection, an allergy, asthma, or a cancer.

A subject having an infection is a subject that has been exposed to aninfectious pathogen and has acute or chronic detectable levels of thepathogen in the body. The CpG immunostimulatory oligonucleotides can beused with or without an antigen to mount an antigen specific systemic ormucosal immune response that is capable of reducing the level of oreradicating the infectious pathogen. An infectious disease, as usedherein, is a disease arising from the presence of a foreignmicroorganism in the body. It is particularly important to developeffective vaccine strategies and treatments to protect the body'smucosal surfaces, which are the primary site of pathogenic entry.

A subject having an allergy is a subject that has or is at risk ofdeveloping an allergic reaction in response to an allergen. An allergyrefers to acquired hypersensitivity to a substance (allergen). Allergicconditions include but are not limited to eczema, allergic rhinitis orcoryza, hay fever, conjunctivitis, bronchial asthma, urticaria (hives)and food allergies, and other atopic conditions.

Allergies are generally caused by IgE antibody generation againstharnless allergens. The cytokines that are induced by systemic ormucosal administration of CpG immunostimulatory oligonucleotides arepredominantly of a class called Th1 (examples are IL-12, IP-10, IFN-αand IFN-γ) and these induce both humoral and cellular immune responses.The other major type of immune response, which is associated with theproduction of IL-4 and IL-5 cytokines, is termed a Th2 immune response.In general, it appears that allergic diseases are mediated by Th2 typeimmune responses. Based on the ability of the CpG immunostimulatoryoligonucleotides to shift the immune response in a subject from apredominant Th2 (which is associated with production of IgE antibodiesand allergy) to a balanced Th2/Th1 response (which is protective againstallergic reactions), an effective dose for inducing an immune responseof a CpG immunostimulatory oligonucleotide can be administered to asubject to treat or prevent asthma and allergy.

Thus, the CpG immunostimulatory oligonucleotides have significanttherapeutic utility in the treatment of allergic and non-allergicconditions such as asthma. Th2 cytokines, especially IL-4 and IL-5 areelevated in the airways of asthmatic subjects. These cytokines promoteimportant aspects of the asthmatic inflammatory response, including IgEisotope switching, eosinophil chemotaxis and activation and mast cellgrowth. Th1 cytokines, especially IFN-γ and IL-12, can suppress theformation of Th2 clones and production of Th2 cytokines. Asthma refersto a disorder of the respiratory system characterized by inflammation,narrowing of the airways and increased reactivity of the airways toinhaled agents. Asthma is frequently, although not exclusivelyassociated with atopic or allergic symptoms.

A subject having a cancer is a subject that has detectable cancerouscells. The cancer may be a malignant or non-malignant cancer. Cancers ortumors include but are not limited to biliary tract cancer; braincancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; intraepithelialneoplasms; lymphomas; liver cancer; lung cancer (e.g. small cell andnon-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer;pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer;testicular cancer; thyroid cancer; and renal cancer, as well as othercarcinomas and sarcomas. In one embodiment the cancer is hairy cellleukemia, chronic myelogenous leukemia, cutaneous T-cell leukemia,multiple myeloma, follicular lymphoma, malignant melanoma, squamous cellcarcinoma, renal cell carcinoma, prostate carcinoma, bladder cellcarcinoma, or colon carcinoma.

A subject shall mean a human or vertebrate animal including but notlimited to a dog, cat, horse, cow, pig, sheep, goat, turkey, chicken,primate, e.g., monkey, and fish (aquaculture species), e.g. salmon.Thus, the invention can also be used to treat cancer and tumors,infections, and allergy/asthma in non human subjects. Cancer is one ofthe leading causes of death in companion animals (i.e., cats and dogs).

As used herein, the term treat, treated, or treating when used withrespect to an disorder such as an infectious disease, cancer, allergy,or asthma refers to a prophylactic treatment which increases theresistance of a subject to development of the disease (e.g., toinfection with a pathogen) or, in other words, decreases the likelihoodthat the subject will develop the disease (e.g., become infected withthe pathogen) as well as a treatment after the subject has developed thedisease in order to fight the disease (e.g., reduce or eliminate theinfection) or prevent the disease from becoming worse.

In the instances when the CpG oligonucleotide is administered with anantigen, the subject may be exposed to the antigen. As used herein, theterm exposed to refers to either the active step of contacting thesubject with an antigen or the passive exposure of the subject to theantigen in vivo. Methods for the active exposure of a subject to anantigen are well-known in the art. In general, an antigen isadministered directly to the subject by any means such as intravenous,intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, orsubcutaneous administration. The antigen can be administeredsystemically or locally. Methods for administering the antigen and theCpG immunostimulatory oligonucleotide are described in more detailbelow. A subject is passively exposed to an antigen if an antigenbecomes available for exposure to the immune cells in the body. Asubject may be passively exposed to an antigen, for instance, by entryof a foreign pathogen into the body or by the development of a tumorcell expressing a foreign antigen on its surface.

The methods in which a subject is passively exposed to an antigen can beparticularly dependent on timing of administration of the CpGimmunostimulatory oligonucleotide. For instance, in a subject at risk ofdeveloping a cancer or an infectious disease or an allergic or asthmaticresponse, the subject may be administered the CpG immunostimulatoryoligonucleotide on a regular basis when that risk is greatest, i.e.,during allergy season or after exposure to a cancer causing agent.Additionally the CpG immunostimulatory oligonucleotide may beadministered to travelers before they travel to foreign lands where theyare at risk of exposure to infectious agents. Likewise the CpGimmunostimulatory oligonucleotide may be administered to soldiers orcivilians at risk of exposure to biowarfare to induce a systemic ormucosal immune response to the antigen when and if the subject isexposed to it.

An antigen as used herein is a molecule capable of provoking an immuneresponse. Antigens include but are not limited to cells, cell extracts,proteins, polypeptides, peptides, polysaccharides, polysaccharideconjugates, peptide and non-peptide mimics of polysaccharides and othermolecules, small molecules, lipids, glycolipids, carbohydrates, virusesand viral extracts and muticellular organisms such as parasites andallergens. The term antigen broadly includes any type of molecule whichis recognized by a host immune system as being foreign. Antigens includebut are not limited to cancer antigens, microbial antigens, andallergens.

A cancer antigen as used herein is a compound, such as a peptide orprotein, associated with a tumor or cancer cell surface and which iscapable of provoking an immune response when expressed on the surface ofan antigen presenting cell in the context of an MHC molecule. Cancerantigens can be prepared from cancer cells either by preparing crudeextracts of cancer cells, for example, as described in Cohen, et al.,1994, Cancer Research, 54:1055, by partially purifying the antigens, byrecombinant technology, or by de novo synthesis of known antigens.Cancer antigens include but are not limited to antigens that arerecombinantly expressed, an immunogenic portion of, or a whole tumor orcancer. Such antigens can be isolated or prepared recombinantly or byany other means known in the art.

A microbial antigen as used herein is an antigen of a microorganism andincludes but is not limited to virus, bacteria, parasites, and fungi.Such antigens include the intact microorganism as well as naturalisolates and fragments or derivatives thereof and also syntheticcompounds which are identical to or similar to natural microorganismantigens and induce an immune response specific for that microorganism.A compound is similar to a natural microorganism antigen if it inducesan immune response (humoral and/or cellular) to a natural microorganismantigen. Such antigens are used routinely in the art and are well knownto those of ordinary skill in the art.

Examples of viruses that have been found in humans include but are notlimited to: Retroviridae (e.g. human immunodeficiency viruses, such asHIV-I (also referred to as HDTV-III, LAVE or HTLV-III/LAV, or HIV-III;and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses,hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses,echoviruses); Calciviridae (e.g. strains that cause gastroenteritis);Togaviridae (e.g. equine encephalitis viruses, rubella viruses);Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow feverviruses); Coronoviridae (e.g. coronaviruses); Rhabdoviradae (e.g.vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebolaviruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus,measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arena viridae (hemorrhagic feverviruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvovirida(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus; Poxyiridae (variola viruses, vaccinia viruses, pox viruses); andIridoviridae (e.g. African swine fever virus); and unclassified viruses(e.g. the agent of delta hepatitis (thought to be a defective satelliteof hepatitis B virus), the agents of non-A, non-B hepatitis (class1=internally transmitted; class 2=parenterally transmitted (i.e.Hepatitis C); Norwalk and related viruses, and astroviruses).

Both gram negative and gram positive bacteria serve as antigens invertebrate animals. Such gram positive bacteria include, but are notlimited to, Pasteurella species, Staphylococci species, andStreptococcus species. Gram negative bacteria include, but are notlimited to, Escherichia coli, Pseudomonas species, and Salmonellaspecies. Specific examples of infectious bacteria include but are notlimited to, Helicobacter pyloris, Borelia burgdorferi, Legionellapneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M.intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus,Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes,Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae(Group B Streptococcus), Streptococcus (viridans group), Streptococcusfaecalis, Streptococcus bovis, Streptococcus (anaerobic sps.),Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcussp., Haemophilus influenzae, Bacillus antracis, corynebacteriumdiphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae,Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes,Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp.,Fusobacterium nucleatum, Streptobacillus moniliformis, Treponemapalladium, Treponema pertenue, Leptospira, Rickettsia, and Actinomycesisraelli.

Examples of fungi include Cryptococcus neoformans, Histoplasmacapsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydiatrachomatis, Candida albicans.

Other infectious organisms (i.e., protists) include Plasmodium spp. suchas Plasmodium falciparum, Plasmodium malariae, Plasmodium ovate, andPlasmodium vivax and Toxoplasma gondii. Blood-borne and/or tissuesparasites include Plasmodium spp., Babesia microti, Babesia divergens,Leishmania tropica, Leishmania spp., Leishmania braziliensis, Leishmaniadonovani, Trypanosoma gambiense and Trypanosoma rhodesiense (Africansleeping sickness), Trypanosoma cruzi (Chagas' disease), and Toxoplasmagondii.

Other medically relevant microorganisms have been described extensivelyin the literature, e.g., see C. G. A Thomas, Medical Microbiology,Bailliere Tindall, Great Britain 1983, the entire contents of which ishereby incorporated by reference.

An allergen refers to a substance (antigen) that can induce an allergicor asthmatic response in a susceptible subject. The list of allergens isenorinous and can include pollens, insect venoms, animal dander dust,fungal spores and drugs (e.g. penicillin). Examples of natural, animaland plant allergens include but are not limited to proteins specific tothe following genuses: Canine (Canis familiaris); Dermatophagoides (e.g.Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia (Ambrosiaartemiisfolia; Lolium (e.g. Lolium perenne or Lolium multiflorum);Cryptomeria (Cryptomeria japonica); Alternaria (Alternaria alternata);Alder; Alnus (Alnus gultinoasa); Betula (Betula verrucosa); Quercus(Quercus alba); Olea (Olea europa); Artemisia (Artemisia vulgaris);Plantago (e.g. Plantago lanceolata); Parietaria (e.g. Parietariaofficinalis or Parietaria judaica); Blattella (e.g. Blattellagermanica); Apis (e.g. Apis multiflorum); Cupressus (e.g. Cupressussempervirens, Cupressus arizonica and Cupressus macrocarpa); Juniperus(e.g. Juniperus sabinoides, Juniperus virginiana, Juniperus communis andJuniperus ashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g.Chamaecyparis obtusa); Periplaneta (e.g. Periplaneta americana);Agropyron (e.g. Agropyron repens); Secale (e.g. Secale cereale);Triticum (e.g. Triticum aestivum); Dactylis (e.g. Dactylis glomerata);Festuca (e.g. Festuca elatior); Poa (e.g. Poapratensis or Poacompressa); Avena (e.g. Avena sativa); Holcus (e.g. Holcus lanatus);Anthoxanthum (e.g. Anthoxanthum odoratum); Arrhenatherum (e.g.Arrhenatherum elatius); Agrostis (e.g. Agrostis alba); Phleum (e.g.Phleum pratense); Phalaris (e.g. Phalaris arundinacea); Paspalum (e.g.Paspalum notatum); Sorghum (e.g. Sorghum halepensis); and Bromus (e.g.Bromus inermis).

The term substantially purified as used herein refers to a polypeptidewhich is substantially free of other proteins, lipids, carbohydrates orother materials with which it is naturally associated. One skilled inthe art can purify viral or bacterial polypeptides using standardtechniques for protein purification. The substantially pure polypeptidewill often yield a single major band on a non-reducing polyacrylamidegel. In the case of partially glycosylated polypeptides or those thathave several start codons, there may be several bands on a non-reducingpolyacrylamide gel, but these will form a distinctive pattern for thatpolypeptide. The purity of the viral or bacterial polypeptide can alsobe determined by amino-terminal amino acid sequence analysis. Othertypes of antigens not encoded by a nucleic acid vector such aspolysaccharides, small molecule, mimics etc are included within theinvention.

The oligonucleotides of the invention may be administered to a subjectwith an anti-microbial agent. An anti-microbial agent, as used herein,refers to a naturally-occurring or synthetic compound which is capableof killing or inhibiting infectious microorganisms. The type ofanti-microbial agent useful according to the invention will depend uponthe type of microorganism with which the subject is infected or at riskof becoming infected. Anti-microbial agents include but are not limitedto anti-bacterial agents, anti-viral agents, anti-fungal agents andanti-parasitic agents. Phrases such as “anti-infective agent”,“anti-bacterial agent”, “anti-viral agent”, “anti-fungal agent”,“anti-parasitic agent” and “parasiticide” have well-established meaningsto those of ordinary skill in the art and are defined in standardmedical texts. Briefly, anti-bacterial agents kill or inhibit bacteria,and include antibiotics as well as other synthetic or natural compoundshaving similar functions. Antibiotics are low molecular weight moleculeswhich are produced as secondary metabolites by cells, such asmicroorganisms. In general, antibiotics interfere with one or morebacterial functions or structures which are specific for themicroorganism and which are not present in host cells. Anti-viral agentscan be isolated from natural sources or synthesized and are useful forkilling or inhibiting viruses. Anti-fungal agents are used to treatsuperficial fungal infections as well as opportunistic and primarysystemic fungal infections. Anti-parasite agents kill or inhibitparasites.

Examples of anti-parasitic agents, also referred to as parasiticidesuseful for human administration include but are not limited toalbendazole, amphotericin B, benznidazole, bithionol, chloroquine HCl,chloroquine phosphate, clindamycin, dehydroemetine, diethylcarbamazine,diloxanide furoate, eflornithine, furazolidaone, glucocorticoids,halofantrine, iodoquinol, ivermectin, mebendazole, mefloquine, meglumineantimoniate, melarsoprol, metrifonate, metronidazole, niclosamide,nifurtimox, oxamniquine, paromomycin, pentamidine isethionate,piperazine, praziquantel, primaquine phosphate, proguanil, pyrantelpamoate, pyrimethanmine-sulfonamides, pyrimethanmine-sulfadoxine,quinacrine HCl, quinine sulfate, quinidine gluconate, spiramycin,stibogluconate sodium (sodium antimony gluconate), suramin,tetracycline, doxycycline, thiabendazole, tinidazole,trimethroprim-sulfamethoxazole, and tryparsamide some of which are usedalone or in combination with others.

Antibacterial agents kill or inhibit the growth or function of bacteria.A large class of antibacterial agents is antibiotics. Antibiotics, whichare effective for killing or inhibiting a wide range of bacteria, arereferred to as broad spectrum antibiotics. Other types of antibioticsare predominantly effective against the bacteria of the classgram-positive or gram-negative. These types of antibiotics are referredto as narrow spectrum antibiotics. Other antibiotics which are effectiveagainst a single organism or disease and not against other types ofbacteria, are referred to as limited spectrum antibiotics. Antibacterialagents are sometimes classified based on their primary mode of action.In general, antibacterial agents are cell wall synthesis inhibitors,cell membrane inhibitors, protein synthesis inhibitors, nucleic acidsynthesis or functional inhibitors, and competitive inhibitors.

Antiviral agents are compounds which prevent infection of cells byviruses or replication of the virus within the cell. There are manyfewer antiviral drugs than antibacterial drugs because the process ofviral replication is so closely related to DNA replication within thehost cell, that non-specific antiviral agents would often be toxic tothe host. There are several stages within the process of viral infectionwhich can be blocked or inhibited by antiviral agents. These stagesinclude, attachment of the virus to the host cell (immunoglobulin orbinding peptides), uncoating of the virus (e.g. amantadine), synthesisor translation of viral mRNA (e.g. interferon), replication of viral RNAor DNA (e.g. nucleotide analogues), maturation of new virus proteins(e.g. protease inhibitors), and budding and release of the virus.

Nucleotide analogues are synthetic compounds which are similar tonucleotides, but which have an incomplete or abnormal deoxyribose orribose group. Once the nucleotide analogues are in the cell, they arephosphorylated, producing the triphosphate formed which competes withnormal nucleotides for incorporation into the viral DNA or RNA. Once thetriphosphate form of the nucleotide analogue is incorporated into thegrowing nucleic acid chain, it causes irreversible association with theviral polymerase and thus chain termination. Nucleotide analoguesinclude, but are not limited to, acyclovir (used for the treatment ofherpes simplex virus and varicella-zoster virus), gancyclovir (usefulfor the treatment of cytomegalovirus), idoxuridine, ribavirin (usefulfor the treatment of respiratory syncitial virus), dideoxyinosine,dideoxycytidine, zidovudine (azidothymidine), imiquimod, andresimiquimod.

The interferons are cytokines which are secreted by virus-infected cellsas well as immune cells. The interferons function by binding to specificreceptors on cells adjacent to the infected cells, causing the change inthe cell which protects it from infection by the virus. α andβ-interferon also induce the expression of Class I and Class II MHCmolecules on the surface of infected cells, resulting in increasedantigen presentation for host immune cell recognition. α andβ-interferons are available as recombinant forms and have been used forthe treatment of chronic hepatitis B and C infection. At the dosageswhich are effective for anti-viral therapy, interferons have severe sideeffects such as fever, malaise and weight loss.

Anti-viral agents useful in the invention include but are not limited toimmunoglobulins, amantadine, interferons, nucleotide analogues, andprotease inhibitors. Specific examples of anti-virals include but arenot limited to Acemannan; Acyclovir; Acyclovir Sodium; Adefovir;Alovudine; Alvircept Sudotox; Amantadine Hydrochloride; Aranotin;Arildone; Atevirdine Mesylate; Avridine; Cidofovir; Cipamfylline;Cytarabine Hydrochloride; Delavirdine Mesylate; Desciclovir; Didanosine;Disoxaril; Edoxudine; Enviradene; Enviroxime; Famciclovir; FamotineHydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscarnet Sodium;Fosfonet Sodium; Ganciclovir; Ganciclovir Sodium; Idoxuridine; Kethoxal;Lamivudine; Lobucavir; Memotine Hydrochloride; Methisazone; Nevirapine;Penciclovir; Pirodavir; Ribavirin; Rimantadine Hydrochloride; SaquinavirMesylate; Somantadine Hydrochloride; Sorivudine; Statolon; Stavudine;Tilorone Hydrochloride; Trifluridine; Valacyclovir Hydrochloride;Vidarabine; Vidarabine Phosphate; Vidarabine Sodium Phosphate; Viroxime;Zalcitabine; Zidovudine; and Zinviroxime.

Anti-fungal agents are useful for the treatment and prevention ofinfective fungi. Anti-fungal agents are sometimes classified by theirmechanism of action. Some anti-fungal agents function as cell wallinhibitors by inhibiting glucose synthase. These include, but are notlimited to, basiungin/ECB. Other anti-fungal agents function bydestabilizing membrane integrity. These include, but are not limited to,immidazoles, such as clotrimazole, sertaconzole, fluconazole,itraconazole, ketoconazole, in iconazole, and voriconacole, as well asFK 463, amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292,butenafine, and terbinafine. Other anti-fungal agents function bybreaking down chitin (e.g. chitinase) or immunosuppression (501 cream).

CpG immunostimulatory oligonucleotides can be combined with othertherapeutic agents such as adjuvants to enhance immune responses. TheCpG immunostimulatory oligonucleotide and other therapeutic agent may beadministered simultaneously or sequentially. When the other therapeuticagents are administered simultaneously they can be administered in thesame or separate formulations, but are administered at the same time.The other therapeutic agents are administered sequentially with oneanother and with CpG immunostimulatory oligonucleotide, when theadministration of the other therapeutic agents and the CpGimmunostimulatory oligonucleotide is temporally separated. Theseparation in time between the administration of these compounds may bea matter of minutes or it may be longer. Other therapeutic agentsinclude but are not limited to adjuvants, cytokines, antibodies,antigens, etc.

The compositions of the invention may also be administered withnon-nucleic acid adjuvants. A non-nucleic acid adjuvant is any moleculeor compound except for the CpG immunostimulatory oligonucleotidesdescribed herein which can stimulate the humoral and/or cellular immuneresponse. Non-nucleic acid adjuvants include, for instance, adjuvantsthat create a depo effect, immune stimulating adjuvants, and adjuvantsthat create a depo effect and stimulate the immune system.

The CpG immunostimulatory oligonucleotides are also useful as mucosaladjuvants. It has previously been discovered that both systemic andmucosal immunity are induced by mucosal delivery of CpG nucleic acids.Thus, the oligonucleotides may be administered in combination with othermucosal adjuvants.

Immune responses can also be induced or augmented by theco-administration or co-linear expression of cytokines (Bueler &Mulligan, 1996; Chow et al., 1997; Geissler et al., 1997; Iwasaki etal., 1997; Kim et al., 1997) or B-7 co-stimulatory molecules (Iwasaki etal., 1997; Tsuji et al., 1997) with the CpG immunostimulatoryoligonucleotides. The term cytokine is used as a generic name for adiverse group of soluble proteins and peptides which act as humoralregulators at nano- to picomolar concentrations and which, either undernormal or pathological conditions, modulate the functional activities ofindividual cells and tissues. These proteins also mediate interactionsbetween cells directly and regulate processes taking place in theextracellular environment. Examples of cytokines include, but are notlimited to IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15,IL-18, granulocyte-macrophage colony stimulating factor (GM-CSF),granulocyte colony stimulating factor (G-CSF), interferon-γ (γ-IFN),IFN-α, tumor necrosis factor (TNF), TGF-β, FLT-3 ligand, and CD40ligand.

The oligonucleotides are also useful for redirecting an immune responsefrom a Th2 immune response to a Th1 immune response. This results in theproduction of a relatively balanced Th1/Th2 environment. Redirection ofan immune response from a Th2 to a Th1 immune response can be assessedby measuring the levels of cytokines produced in response to the nucleicacid (e.g., by inducing monocytic cells and other cells to produce Th1cytokines, including IL-12, IFN-γ and GM-CSF). The redirection orrebalance of the immune response from a Th2 to a Th1 response isparticularly useful for the treatment or prevention of asthma. Forinstance, an effective amount for treating asthma can be that amount;useful for redirecting a Th2 type of immune response that is associatedwith asthma to a Th1 type of response or a balanced Th1/Th2 environment.Th2 cytokines, especially IL-4 and IL-5 are elevated in the airways ofasthmatic subjects. The CpG immunostimulatory oligonucleotides of theinvention cause an increase in Th1 cytokines which helps to rebalancethe immune system, preventing or reducing the adverse effects associatedwith a predominately Th2 immune response.

The oligonucleotides of the invention may also be useful for treatingairway remodeling. Airway remodeling results from smooth muscle cellproliferation and/or submucosal thickening in the airways, andultimately causes narrowing of the airways leading to restrictedairflow. The oligonucleotides of the invention may prevent furtherremodeling and possibly even reduce tissue build up resulting from theremodeling process.

The oligonucleotides are also useful for improving survival,differentiation, activation and maturation of dendritic cells. The CpGimmunostimulatory oligonucleotides have the unique capability to promotecell survival, differentiation, activation and maturation of dendriticcells.

CpG immunostimulatory oligonucleotides also increase natural killer celllytic activity and antibody dependent cellular cytotoxicity (ADCC). ADCCcan be performed using a CpG immunostimulatory oligonucleotide incombination with an antibody specific for a cellular target, such as acancer cell. When the CpG immunostimulatory oligonucleotide isadministered to a subject in conjunction with the antibody the subject'simmune system is induced to kill the tumor cell. The antibodies usefulin the ADCC procedure include antibodies which interact with a cell inthe body. Many such antibodies specific for cellular targets have beendescribed in the art and many are commercially available.

The CpG immunostimulatory oligonucleotides may also be administered inconjunction with an anti-cancer therapy. Anti-cancer therapies includecancer medicaments, radiation and surgical procedures. As used herein, a“cancer medicament” refers to a agent which is administered to a subjectfor the purpose of treating a cancer. As used herein, “treating cancer”includes preventing the development of a cancer, reducing the symptomsof cancer, and/or inhibiting the growth of an established cancer. Inother aspects, the cancer medicament is administered to a subject atrisk of developing a cancer for the purpose of reducing the risk ofdeveloping the cancer. Various types of medicaments for the treatment ofcancer are described herein. For the purpose of this specification,cancer medicaments are classified as chemotherapeutic agents,immunotherapeutic agents, cancer vaccines, hormone therapy, andbiological response modifiers.

Additionally, the methods of the invention are intended to embrace theuse of more than one cancer medicament along with the CpGimmunostimulatory oligonucleotides. As an example, where appropriate,the CpG immunostimulatory oligonucleotides may be administered with botha chemotherapeutic agent and an immunotherapeutic agent. Alternatively,the cancer medicament may embrace an immunotherapeutic agent and acancer vaccine, or a chemotherapeutic agent and a cancer vaccine, or achemotherapeutic agent, an immunotherapeutic agent and a cancer vaccineall administered to one subject for the purpose of treating a subjecthaving a cancer or at risk of developing a cancer.

The chemotherapeutic agent may be selected from the group consisting ofmethotrexate, vincristine, adriamycin, cisplatin, non-sugar containingchloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin,doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin,carmustaine and poliferposan, MM1270, BAY 12-9566, RAS farnesyltransferase inhibitor, farnesyl transferase inhibitor, MMP,MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470,Hycamtin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone,Metaret/Suramin, Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340,AG3433, Incel/VX-710, VX-853, ZD0101, IS1641, ODN 698, TA2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX8951f,Lemonal DP 2202, FK 317, Picibanil/OK-432, AD 32/Valrubicin,Metastron/strontium derivative, Temodal/Temozolom ide, Evacet/liposomaldoxorubicin, Yewtaxan/Paclitaxel, Taxol/Paclitaxel, Xeload/Capecitabine,Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid,SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609(754)/RAS oncogene inhibitor, BMS-182751/oral platinum, UFT(Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/776C85/5FU enhancer,Campto/Levamisole, Camptosar/Irinotecan, Tumodex/Ralitrexed,Leustatin/Cladribine, Paxex/Paclitaxel, Doxil/liposomal doxorubicin,Caelyx/liposomal doxorubicin, Fludara/Fludarabine,Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU 79553/Bis-Naphtalimide, LU103793/Dolastain, Caetyx/liposomal doxorubicin, Gemzar/Geincitabine, ZD0473/Anormed, YM 116, Iodine seeds, CDK4 and CDK2 inhibitors, PARPinhibitors, D4809/Dexifosamide, Ifes/Mesnex/Ifosamide, Vumon/Teniposide,Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD9331, Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane Analog,nitrosoureas, alkylating agents such as melphelan and cyclophosphamide,Aminoglutethimide, Asparaginase, Busulfan, Carboplatin, Chlorombucil,Cytarabine HCl, Dactinomycin, Daunorubicin HCl, Estramustine phosphatesodium, Etoposide (VP16-213), Floxuridine, Fluorouracil (5-FU),Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfaimide, InterferonAlfa-2a, Alfa-2b, Leuprolide acetate (LHRH-releasing factor analogue),Lomustine (CCNU), Mechlorethamine HCl (nitrogen mustard),Mercaptopurine, Mesna, Mitotane (o.p′-DDD), Mitoxantrone HCl,Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifencitrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m-AMSA),Azacitidine, Erthropoietin, Hexamethylmelamine (HMM), Interleukin 2,Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG),Pentostatin (2′deoxycoformycin), Semustine (methyl-CCNU), Teniposide(VM-26) and Vindesine sulfate, but it is not so limited.

The immunotherapeutic agent may be selected from the group consisting ofRibutaxin, Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225,Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, LDP-03, ior t6, MDX-210,MDX-11, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447,MELIMMUNE-2, MELIMMUNE-1, CEACIDE, Pretarget, NovoMAb-G2, TNT,Gliomab-H, GNI-250, EMD-72000, LymphoCide, CMA 676, Monopharm-C, 4B5,ior egf.r3, ior c5, BABS, anti-FLK-2, MDX-260, ANA Ab, SMART ID10 Ab,SMART ABL 364 Ab and ImmuRAIT-CEA, but it is not so limited.

The cancer vaccine may be selected from the group consisting of EGF,Anti-idiotypic cancer vaccines, Gp75 antigen, GMK melanoma vaccine, MGVganglioside conjugate vaccine, Her2/neu, Ovarex, M-Vax, O-Vax, L-Vax,STn-KHL theratope, BLP25 (MUC-1), liposomal idiotypic vaccine, Melacine,peptide antigen vaccines, toxin/antigen vaccines, MVA-based vaccine,PACIS, BCG vacine, TA-HPV, TA-CIN, DISC-virus and ImmuCyst/TheraCys, butit is not so limited.

The use of CpG immunostimulatory oligonucleotides in conjunction withimmunotherapeutic agents such as monoclonal antibodies is able toincrease long-term survival through a number of mechanisms includingsignificant enhancement of ADCC (as discussed above), activation ofnatural killer (NK) cells and an increase in IFNα levels. The nucleicacids when used in combination with monoclonal antibodies serve toreduce the dose of the antibody required to achieve a biological result.

As used herein, the terms “cancer antigen” and “tumor antigen” are usedinterchangeably to refer to antigens which are differentially expressedby cancer cells and can thereby be exploited in order to target cancercells. Cancer antigens are antigens which can potentially stimulateapparently tumor-specific immune responses. Some of these antigens areencoded, although not necessarily expressed, by normal cells. Theseantigens can be characterized as those which are normally silent (i.e.,not expressed) in normal cells, those that are expressed only at certainstages of differentiation and those that are temporally expressed suchas embryonic and fetal antigens. Other cancer antigens are encoded bymutant cellular genes, such as oncogenes (e.g., activated ras oncogene),suppressor genes (e.g., mutant p53), fusion proteins resulting frominternal deletions or chromosomal translocations. Still other cancerantigens can be encoded by viral genes such as those carried on RNA andDNA tumor viruses.

The CpG immunostimulatory oligonucleotides are also useful for treatingand prevneting autoimmune disease. Autoimmune disease is a class ofdiseases in which an subject's own antibodies react with host tissue orin which immune effector T cells are autoreactive to endogenous selfpeptides and cause destruction of tissue. Thus an immune response ismounted against a subject's own antigens, referred to as self antigens.Autoimmune diseases include but are not limited to rheumatoid arthritis,Crohn's disease, multiple sclerosis, systemic lupus erythematosus (SLE),autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto'sthyroiditis, Goodpasture's syndrome, pemphigus (e.g., pemphigusvulgaris), Grave's disease, autoimmune hemolytic anemia, autoimmunethrombocytopenic purpura, scleroderma with anti-collagen antibodies,mixed connective tissue disease, polymyositis, pernicious anemia,idiopathic Addison's disease, autoimmune-associated infertility,glomerulonephritis (e.g., crescentic glomerulonephritis, proliferativeglomerulonephritis), bullous pemphigoid, Sjögren's syndrome, insulinresistance, and autoimmune diabetes mellitus.

A “self-antigen” as used herein refers to an antigen of a normal hosttissue. Normal host tissue does not include cancer cells. Thus an immuneresponse mounted against a self-antigen, in the context of an autoimmunedisease, is an undesirable immune response and contributes todestruction and damage of normal tissue, whereas an immune responsemounted against a cancer antigen is a desirable immune response andcontributes to the destruction of the tumor or cancer. Thus, in someaspects of the invention aimed at treating autoimmune disorders it isnot recommended that the CpG immunostimulatory nucleic acids beadministered with self antigens, particularly those that are the targetsof the autoimmune disorder.

In other instances, the the CpG immunostimulatory nucleic acids may bedelivered with low doses of self-antigens. A number of animal studieshave demonstrated that mucosal administration of low doses of antigencan result in a state of immune hyporesponsiveness or “tolerance.” Theactive mechanism appears to be a cytokine-mediated immune deviation awayfrom a Th1 towards a predominantly Th2 and Th3 (i.e., TGF-β dominated)response. The active suppression with low dose antigen delivery can alsosuppress an unrelated immune response (bystander suppression) which isof considerable interest in the therapy of autoimmune diseases, forexample, rheumatoid arthritis and SLE. Bystander suppression involvesthe secretion of Th1-counter-regulatory, suppressor cytokines in thelocal environment where proinflammatory and Th1 cytokines are releasedin either an antigen-specific or antigen-nonspecific manner. “Tolerance”as used herein is used to refer to this phenomenon. Indeed, oraltolerance has been effective in the treatment of a number of autoimmunediseases in animals including: experimental autoimmune encephalomyelitis(EAE), experimental autoimmune myasthenia gravis, collagen-inducedarthritis (CIA), and insulin-dependent diabetes mellitus. In thesemodels, the prevention and suppression of autoimmune disease isassociated with a shift in antigen-specific humoral and cellularresponses from a Th1 to Th2/Th3 response.

The invention also includes a method for inducing antigen non-specificinnate immune activation and broad spectrum resistance to infectiouschallenge using the CpG immunostimulatory oligonucleotides. The termantigen non-specific innate immune activation as used herein refers tothe activation of immune cells other than B cells and for instance caninclude the activation of NK cells, T cells or other immune cells thatcan respond in an antigen independent fashion or some combination ofthese cells. A broad spectrum resistance to infectious challenge isinduced because the immune cells are in active form and are primed torespond to any invading compound or microorganism. The cells do not haveto be specifically primed against a particular antigen. This isparticularly useful in biowarfare, and the other circumstances describedabove such as travelers.

The invention also relates to oligonucleotides having chiralinternucleotide linkages. As described above the soft and semi-softoligonucleotides of the invention may have phosphodiester like linkagesbetween C and G. One example of a phosphodiester-like linkage is aphosphorothioate linkage in an Rp conformation.

At least one study has examined the effect of p-chirality upon theimmune stimulatory effects of CpG oligonucleotides. Yu et al., comparedstereo-enriched (not stereo-pure) phosphorothioate (PS)-oligonucleotidesfor their ability to induce spleen cell proliferation (Yu et al., 2000).In that study, a 19mer sequence containing a single CpG motif was foundto induce high levels of mouse spleen cell proliferation if theoligonucleotide was synthesized with random p-chirality or was enrichedfor Sp internucleotide linkages, but the proliferation was markedlyreduced if the oligonucleotide was enriched for Rp internucleotidelinkages (Yu et al., 2000). However, that study did not examine thespecific role of p-chirality at the CpG dinucleotide, nor did itdetermine whether the Rp CpG oligonucleotides would have activity inshort term stimulation assays.

It has been discovered according to the invention that oligonucleotidep-chirality can have apparently opposite effects on the immune activityof a CpG oligonucleotide, depending upon the time point at whichactivity is measured. At an early time point of 40 minutes, the R_(p)but not the S_(p) stereoisomer of phosphorothioate CpG oligonucleotideinduces JNK phosphorylation in mouse spleen cells (discussed inexamples). In contrast, when assayed at a late time point of 44 hr, theS_(p) but not the R_(p) stereoisomer is active in stimulating spleencell proliferation. We have demonstrated that this difference in thekinetics and bioactivity of the R_(p) and S_(p) stereoisomers does notresult from any difference in cell uptake, but rather most likely is dueto two opposing biologic roles of the p-chirality. First, the enhancedactivity of the Rp stereoisomer compared to the Sp for stimulatingimmune cells at early time points indicates that the Rp may be moreeffective at interacting with the CpG receptor, TLR9, or inducing thedownstream signaling pathways. On the other hand, the faster degradationof the Rp PS-oligonucleotides compared to the Sp results in a muchshorter duration of signaling, so that the Sp PS-oligonucleotides appearto be more biologically active when tested at later time points.

The invention in some aspects is based on the novel finding that thepreviously reported relative lack of immune stimulation by Rp PS-Oligosis due only to their nuclease lability, not to an inherent inability tostimulate the CpG receptor and downstream pathways. When tested fortheir ability to stimulate JNK phosphorylation, which indicatesactivation of this mitogen activated protein kinase pathway, the Rpoligonucleotide appeared to be the most active, followed by thestereo-random oligo, but with no detectable activity of the Spoligonucleotide. However, when these oligonucleotide were compared fortheir ability to activate the NF-κB pathway, as measured by thedegradation of the inhibitory protein IκB-α, all of the CpGoligonucleotide were active, although the non-CpG control failed toinduce IκB-α degradation. Thus, the Sp oligonucleotide is stillbiologically active. It's failure to induce the JNK pathway could berelated to differences in the kinetics of activation of the JNK andNF-κB pathways, but due to limited amounts of the stereo-specificoligonucleotide that were available for testing, we were unable toconfirm this hypothesis.

The experiments described in the Examples revealed a surprisingly strongeffect of the p-chirality at the CpG dinucleotide itself. In comparisonto a stereo-random CpG oligonucleotide the congener in which the singleCpG dinucleotide was linked in Rp was slightly more active, while thecongener containing an Sp linkage was nearly inactive for inducingspleen cell proliferation. The loss of activity of the Sp congenersupports our hypothesis that the TLR9 receptor may not be indifferent tothe chirality of the CpG dinucleotide in the DNA with which itinteracts, but may actually be stimulated better by the Rp stereoisomer.Thus, the stimulatory effect of the stereo-random oligo is probably notonly due to the presence of 50% Sp linkages that retard degradation, butalso to the fact that half of the oligo molecules will have Rp chiralityat the CpG dinucleotide, which appears to enhance the immune stimulatoryeffects.

The nuclease sensitivity of R_(p) PS linkages has important implicationsfor interpretation of pharmacokinetic (PK) and metabolism studies ofPS-Oligos in humans or animals. The predominant serum nuclease activityis known to be a 3′ exonuclease. In a typical stereo-random PS-oligosolution the last 3′ internucleotide linkage will be expected to be ofR_(p) chirality in one half of the molecules. Therefore in these 50% ofthe PS-Oligo molecules, the terminal 3′ base will be cleaved fairlyrapidly after IV infusion. The second from the end 3′ internucleotidelinkage should be of R_(p) chirality in one half of these molecules, andtherefore in 25% of the starting PS-Oligo molecules the 3′ end may beexpected to be shortened by 2 bases relatively rapidly. This in vivobase-clipping process involving the 3′ R_(p) internucleotide linkagesmay be expected to continue until the 3′ internucleotide linkage is ofSp configuration. Therefore, if the PS-Oligos were synthesized to havean Sp 3′ terminal linkage, they should have much slower degradation anda different PK profile compared to stereo-random PS-Oligos. This shouldmake it possible to use somewhat shorter oligonucleotide for in vivoapplications. In designing optimized oligos for antisense applications,the enhanced RNA binding of the Rp stereoisomer points to thedesirability of having as much of the internal core of theoligonucleotide in Rp configuration as possible. On the other hand, anoptimized CpG oligonucleotide for immunostimulatory applications may beone in which all of the internucleotide linkages except the CpG would beof Sp chirality.

The CpG immunostimulatory oligonucleotides may be directly administeredto the subject or may be administered in conjunction with a nucleic aciddelivery complex. A nucleic acid delivery complex shall mean a nucleicacid molecule associated with (e.g. ionically or covalently bound to; orencapsulated within) a targeting means (e.g. a molecule that results inhigher affinity binding to target cell. Examples of nucleic aciddelivery complexes include nucleic acids associated with a sterol (e.g.cholesterol), a lipid (e.g. a cationic lipid, virosome or liposome), ora target cell specific binding agent (e.g. a ligand recognized by targetcell specific receptor). Preferred complexes may be sufficiently stablein vivo to prevent significant uncoupling prior to internalization bythe target cell. However, the complex can be cleavable under appropriateconditions within the cell so that the oligonucleotide is released in afunctional form.

Delivery vehicles or delivery devices for delivering antigen andoligonucleotides to surfaces have been described. The CpGimmunostimulatory oligonucleotide and/or the antigen and/or othertherapeutics may be administered alone (e.g., in saline or buffer) orusing any delivery vehicles known in the art. For instance the followingdelivery vehicles have been described: Cochleates (Gould-Fogerite etal., 1994, 1996); Emulsomes (Vancott et al., 1998, Lowell et al., 1997);ISCOMs (Mowat et al., 1993, Carlsson et al., 1991, Hu et., 1998, Moreinet al., 1999); Liposomes (Childers et al., 1999, Michalek et al., 1989,1992, de Haan 1995a, 1995b); Live bacterial vectors (e.g., Salmonella,Escherichia coli, Bacillus calmatte-guerin, Shigella, Lactobacillus)(Hone et al., 1996, Pouwels et al., 1998, Chatfield et al., 1993, Stoveret al., 1991, Nugent et al., 1998); Live viral vectors (e.g., Vaccinia,adenovirus, Herpes Simplex) (Gallichan et al., 1993, 1995, Moss et al.,1996, Nugent et al., 1998, Flexner et al., 1988, Morrow et al., 1999);Microspheres (Gupta et al., 1998, Jones et al., 1996, Maloy et al.,1994, Moore et al., 1995, O'Hagan et al., 1994, Eldridge et al., 1989);Nucleic acid vaccines (Fynan et al., 1993, Kuklin et al., 1997, Sasakiet al., 1998, Okada et al., 1997, Ishii et al., 1997); Polymers (e.g.carboxymethylcellulose, chitosan) (Hamajima et al., 1998, Jabbal-Gill etal., 1998); Polymer rings (Wyatt et al., 1998); Proteosomes (Vancott etal., 1998, Lowell et al., 1988, 1996, 1997); Sodium Fluoride (Hashi etal., 1998); Transgenic plants (Tacket et al., 1998, Mason et al., 1998,Haq et al., 1995); Virosomes (Gluck et al., 1992, Mengiardi et al.,1995, Cryz et al., 1998); Virus-like particles (Jiang et al., 1999,Leibl et al., 1998). Other delivery vehicles are known in the art andsome additional examples are provided below in the discussion ofvectors.

The term effective amount of a CpG immunostimulatory oligonucleotiderefers to the amount necessary or sufficient to realize a desiredbiologic effect. For example, an effective amount of a CpGimmunostimulatory oligonucleotide administered with an antigen forinducing mucosal immunity is that amount necessary to cause thedevelopment of IgA in response to an antigen upon exposure to theantigen, whereas that amount required for inducing systemic immunity isthat amount necessary to cause the development of IgG in response to anantigen upon exposure to the antigen. Combined with the teachingsprovided herein, by choosing among the various active compounds andweighing factors such as potency, relative bioavailability, patient bodyweight, severity of adverse side-effects and preferred mode ofadministration, an effective prophylactic or therapeutic treatmentregimen can be planned which does not cause substantial toxicity and yetis entirely effective to treat the particular subject. The effectiveamount for any particular application can vary depending on such factorsas the disease or condition being treated, the particular CpGimmunostimulatory oligonucleotide being administered the size of thesubject, or the severity of the disease or condition. One of ordinaryskill in the art can empirically determine the effective amount of aparticular CpG immunostimulatory oligonucleotide and/or antigen and/orother therapeutic agent without necessitating undue experimentation.

Subject doses of the compounds described herein for mucosal or localdelivery typically range from about 0.1 μg to 10 mg per administration,which depending on the application could be given daily, weekly, ormonthly and any other amount of time therebetween. More typicallymucosal or local doses range from about 10 μg to 5 mg peradministration, and most typically from about 100 μg to 1 mg, with 2-4administrations being spaced days or weeks apart. More typically, immunestimulant doses range from 1 μg to 10 mg per administration, and mosttypically 10 μg to 1 mg, with daily or weekly administrations. Subjectdoses of the compounds described herein for parenteral delivery for thepurpose of inducing an antigen-specific immune response, wherein thecompounds are delivered with an antigen but not another therapeuticagent are typically 5 to 10,000 times higher than the effective mucosaldose for vaccine adjuvant or immune stimulant applications, and moretypically 10 to 1,000 times higher, and most typically 20 to 100 timeshigher. Doses of the compounds described herein for parenteral deliveryfor the purpose of inducing an innate immune response or for increasingADCC or for inducing an antigen specific immune response when the CpGimmunostimulatory oligonucleotides are administered in combination withother therapeutic agents or in specialized delivery vehicles typicallyrange from about 0.1 μg to 10 mg per administration, which depending onthe application could be given daily, weekly, or monthly and any otheramount of time therebetween. More typically parenteral doses for thesepurposes range from about 10 μg to 5 mg per administration, and mosttypically from about 100 μg to 1 mg, with 2-4 administrations beingspaced days or weeks apart. In some embodiments, however, parenteraldoses for these purposes may be used in a range of 5 to 10,000 timeshigher than the typical doses described above.

For any compound described herein the therapeutically effective amountcan be initially determined from animal models. A therapeuticallyeffective dose can also be determined from human data for CpGoligonucleotides which have been tested in humans (human clinical trialshave been initiated) and for compounds which are known to exhibitsimilar pharmacological activities, such as other adjuvants, e.g., LTand other antigens for vaccination purposes. Higher doses may berequired for parenteral administration. The applied dose can be adjustedbased on the relative bioavailability and potency of the administeredcompound. Adjusting the dose to achieve maximal efficacy based on themethods described above and other methods as are well-known in the artis well within the capabilities of the ordinarily skilled artisan.

The formulations of the invention are administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, adjuvants, and optionally other therapeuticingredients.

For use in therapy, an effective amount of the CpG immunostimulatoryoligonucleotide can be administered to a subject by any mode thatdelivers the oligonucleotide to the desired surface, e.g., mucosal,systemic. Administering the pharmaceutical composition of the presentinvention may be accomplished by any means known to the skilled artisan.Preferred routes of administration include but are not limited to oral,parenteral, intramuscular, intranasal, sublingual, intratracheal,inhalation, ocular, vaginal, and rectal.

For oral administration, the compounds (i.e., CpG immunostimulatoryoligonucleotides, antigens and other therapeutic agents) can beformulated readily by combining the active compound(s) withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the compounds of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject to be treated.Pharmaceutical preparations for oral use can be obtained as solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate. Optionally the oral formulations may also be formulated insaline or buffers, i.e. EDTA for neutralizing internal acid conditionsor may be administered without any carriers.

Also specifically contemplated are oral dosage forms of the abovecomponent or components. The component or components may be chemicallymodified so that oral delivery of the derivative is efficacious.Generally, the chemical modification contemplated is the attachment ofat least one moiety to the component molecule itself, where said moietypermits (a) inhibition of proteolysis; and (b) uptake into the bloodstream from the stomach or intestine. Also desired is the increase inoverall stability of the component or components and increase incirculation time in the body. Examples of such moieties include:polyethylene glycol, copolymers of ethylene glycol and propylene glycol,carboxym ethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone and polyproline. Abuchowski and Davis, 1981, “SolublePolymer-Enzyme Adducts” In: Enzymes as Drugs, Hocenberg and Roberts,eds., Wiley-Interscience, New York, N.Y., pp. 367-383; Newmark, et al.,1982, J. Appl. Biochem. 4:185-189. Other polymers that could be used arepoly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred forpharmaceutical usage, as indicated above, are polyethylene glycolmoieties.

For the component (or derivative) the location of release may be thestomach, the small intestine (the duodenum, the jejunum, or the ileum),or the large intestine. One skilled in the art has availableformulations which will not dissolve in the stomach, yet will releasethe material in the duodenum or elsewhere in the intestine. Preferably,the release will avoid the deleterious effects of the stomachenvironment, either by protection of the oligonucleotide (or derivative)or by release of the biologically active material beyond the stomachenvironment, such as in the intestine.

To ensure full gastric resistance a coating impermeable to at least pH5.0 is essential. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings which make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic i.e. powder; for liquid forms, a soft gelatin shell may beused. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, molded tablets or tablet triturates,moist massing techniques can be used.

The therapeutic can be included in the formulation as finemulti-particulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, theoligonucleotide (or derivative) may be formulated (such as by liposomeor microsphere encapsulation) and then further contained within anedible product, such as a refrigerated beverage containing colorants andflavoring agents.

One may dilute or increase the volume of the therapeutic with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrates include but are notlimited to starch, including the commercial disintegrant based onstarch, Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. Another form of the disintegrants are the insolublecationic exchange resins. Powdered gums may be used as disintegrants andas binders and these can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders may be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Others include methyl cellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An anti-frictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment asurfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents mightbe used and could include benzalkonium chloride or benzethomiumchloride. The list of potential non-ionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the oligonucleotideor derivative either alone or as a mixture in different ratios.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsulator may be formulated containing a powder mix of the compound anda suitable powder base such as lactose or starch.

Also contemplated herein is pulmonary delivery of the oligonucleotides(or derivatives thereof). The oligonucleotide (or derivative) isdelivered to the lungs of a mammal while inhaling and traverses acrossthe lung epithelial lining to the blood stream. Other reports of inhaledmolecules include Adjei et al., 1990, Pharmaceutical Research,7:565-569; Adjei et al., 1990, International Journal of Pharmaceutics,63:135-144 (leuprolide acetate); Braquet et al., 1989, Journal ofCardiovascular Pharmacology, 13(suppl. 5):143-146 (endothelin-1);Hubbard et al., 1989, Annals of Internal Medicine, Vol. III, pp. 206-212(al-antitrypsin); Smith et al., 1989, J. Clin. Invest. 84:1145-1146(a-1-proteinase); Oswein et al., 1990, “Aerosolization of Proteins”,Proceedings of Symposium on Respiratory Drug Delivery II, Keystone,Colo., March, (recombinant human growth hormone); Debs et al., 1988, J.Immunol. 140:3482-3488 (interferon-g and tumor necrosis factor alpha)and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colonystimulating factor). A method and composition for pulmonary delivery ofdrugs for systemic effect is described in U.S. Pat. No. 5,451,569,issued Sep. 19, 1995 to Wong et al.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art.

Some specific examples of commercially available devices suitable forthe practice of this invention are the Ultravent nebulizer, manufacturedby Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer,manufactured by Marquest Medical Products, Englewood, Colo.; theVentolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; and the Spinhaler powder inhaler, manufactured byFisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of oligonucleotide (or derivative). Typically, eachformulation is specific to the type of device employed and may involvethe use of an appropriate propellant material, in addition to the usualdiluents, adjuvants and/or carriers useful in therapy. Also, the use ofliposomes, microcapsules or microspheres, inclusion complexes, or othertypes of carriers is contemplated. Chemically modified oligonucleotidemay also be prepared in different formulations depending on the type ofchemical modification or the type of device employed.

Formulations suitable for use with a nebulizer, eitherjet or ultrasonic,will typically comprise oligonucleotide (or derivative) dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeoligonucleotide per mL of solution. The formulation may also include abuffer and a simple sugar (e.g., for oligonucleotide stabilization andregulation of osmotic pressure). The nebulizer formulation may alsocontain a surfactant, to reduce or prevent surface induced aggregationof the oligonucleotide caused by atomization of the solution in formingthe aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the oligonucleotide (orderivative) suspended in a propellant with the aid of a surfactant. Thepropellant may be any conventional material employed for this purpose,such as a chlorofluorocarbon, a hydrochlorofluorocarbon, ahydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol, and1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactantsinclude sorbitan trioleate and soya lecithin. Oleic acid may also beuseful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing oligonucleotide (or derivative) andmay also include a bulking agent, such as lactose, sorbitol, sucrose, ormannitol in amounts which facilitate dispersal of the powder from thedevice, e.g., 50 to 90% by weight of the formulation. Theoligonucleotide (or derivative) should most advantageously be preparedin particulate form with an average particle size of less than 10 mm (ormicrons), most preferably 0.5 to 5 mm, for most effective delivery tothe distal lung.

Nasal delivery of a pharmaceutical composition of the present inventionis also contemplated. Nasal delivery allows the passage of apharmaceutical composition of the present invention to the blood streamdirectly after administering the therapeutic product to the nose,without the necessity for deposition of the product in the lung.Formulations for nasal delivery include those with dextran orcyclodextran.

For nasal administration, a useful device is a small, hard bottle towhich a metered dose sprayer is attached. In one embodiment, the metereddose is delivered by drawing the pharmaceutical composition of thepresent invention solution into a chamber of defined volume, whichchamber has an aperture dimensioned to aerosolize and aerosolformulation by forming a spray when a liquid in the chamber iscompressed. The chamber is compressed to administer the pharmaceuticalcomposition of the present invention. In a specific embodiment, thechamber is a piston arrangement. Such devices are commerciallyavailable.

Alternatively, a plastic squeeze bottle with an aperture or openingdimensioned to aerosolize an aerosol formulation by forming a spray whensqueezed is used. The opening is usually found in the top of the bottle,and the top is generally tapered to partially fit in the nasal passagesfor efficient administration of the aerosol formulation. Preferably, thenasal inhaler will provide a metered amount of the aerosol formulation,for administration of a measured dose of the drug.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer, Science 249:1527-1533,1990, which is incorporated herein by reference.

The CpG immunostimulatory oligonucleotides and optionally othertherapeutics and/or antigens may be administered per se (neat) or in theform of a pharmaceutically acceptable salt. When used in medicine thesalts should be pharmaceutically acceptable, but non-pharmaceuticallyacceptable salts may conveniently be used to prepare pharmaceuticallyacceptable salts thereof. Such salts include, but are not limited to,those prepared from the following acids: hydrochloric, hydrobromic,sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulphonic, tartaric, citric, methane sulphonic, formic, malonic,succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, suchsalts can be prepared as alkaline metal or alkaline earth salts, such assodium, potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

The pharmaceutical compositions of the invention contain an effectiveamount of a CpG immunostimulatory oligonucleotide and optionallyantigens and/or other therapeutic agents optionally included in apharmaceutically-acceptable carrier. The termpharmaceutically-acceptable carrier means one or more compatible solidor liquid filler, diluents or encapsulating substances which aresuitable for administration to a human or other vertebrate animal. Theterm carrier denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being commingled with the compounds of the presentinvention, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficiency.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES

Materials and Methods:

Oligodeoxynucleotides (ODNs)

All ODNs were purchased from biospring (Frankfurt, Germany) or Sigma-Ark(Darmstadt, Germany), and were controlled for identity and purity byColey Pharmaceutical GmbH (Langenfeld, Germany). ODNs were diluted inphosphate-buffered saline (Sigma, Germany), and stored at −20° C. Alldilutions were carried out using pyrogen-free reagents.

Cell Purification

Peripheral blood buffy coat preparations from healthy male and femalehuman donors were obtained from the Blood Bank of the University ofDüsseldorf (Germany) and from these, PBMC were purified bycentrifugation over Ficoll-Hypaque (Sigma). The purified PBMC wereeither used freshly (for most assays) or were suspended in freezingmedium and stored at −70° C. When required, aliquots of these cells werethawed, washed and resuspended in RPMI 1640 culture medium(BioWhittaker, Belgium) supplemented with 5% (v/v) heat inactivatedhuman AB serum (BioWhittaker, Belgium) or 10% (v/v) heat inactivatedFCS, 2 mM L-glutamine (BioWhittaker), 100 U/ml penicillin and 100 μg/mlstreptomycin (Invitrogen (Karlsruhe, Germany)).

Cytokine Detection

Thawed or fresh PBMC were seeded on 48 well flat-bottom plates, or 96well round-bottom plates, and incubated with ODN in the concentrationsas indicated in a humidified incubator at 37° C. Culture supernatantswere collected and if not used immediately, were frozen at −20° C. untilrequired. Amounts of cytokines in the supernatants were assessed usingcommercially available ELISA Kits (Diaclone, USA) or in-house ELISAsdeveloped using commercially available antibodies (from BectonDickinson/Pharmingen or PBL).

Cultures for Flow Cytometric Analysis of NK Cell Activation

Fluorochrome conjugated monoclonal antibodies to CD3 (T cell marker),CD56 (NK cell marker) and CD69 (early activation marker on NK cells andT cells) were purchased from Becton Dickinson. PBMC were incubated for24 hours with or without the addition of different concentrations ofODNs in 96 well round-bottom plates. NK cells were identified asCD56-positive and CD3-negative cells by flow cytometry. Flow cytometricdata were acquired on a FACSCalibur (Becton Dickinson). Data wereanalyzed using the computer program CellQuest (Becton Dickinson).

Flow Cytometric Analysis of Cell Surface Activation Markers

For measurement of the expression of the co-stimulatory molecule CD86 asan activation marker on B cells, PBMCs were incubated for 48 h with ODNin the concentrations as indicated, and cells were stained with mAb forCD19 and CD86 (Pharmingen, Germany). CD86 expression on CD19 positive Bcells was measured by flow cytometry.

For measurement of the expression of the co-stimulatory molecule CD80 asan activation marker on monocytes, PBMCs were incubated for 48 h withODN in the concentrations as indicated, and cells were stained with mAbfor CD14, CD19, and CD80 (Pharmingen, Germany). CD80 expression on CD14positive CD19 negative monocytes was measured by flow cytometry. Theresults of both measurements are given as Mean Fluorescence Intensity(MFI).

Example 1

Levels of interferon-alpha (IFN-α), IFN-γ, IL-10, IL-6, and TNF-αsecreted from human PBMC following exposure of these cells to the CpGoligonucleotides described herein is shown in the attached FIGS. 1-5.The test oligonucleotides examined are depicted in the figures by a ▴.An oligonucleotide that served as a positive control oligonucleotide wasdepicted by a ▪. The test oligonucleotides shown in FIGS. 1A, 2A, 3A,4A, and 5A include SEQ ID NO: 322, SEQ ID NO: 323, and SEQ ID NO: 324The test oligonucleotides shown in FIGS. 1B, 2B, 3B, 4B, and 5B includeSEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, and SEQ ID NO: 328. Theconcentration of oligonucleotide used to produce a particular data pointis depicted along the X-axis (μM). Below the graphs the level ofcytokine secreted by cells treated with a negative (medium) and in somecases LPS is listed for each experiment.

As demonstrated in FIGS. 1-5 the oligonucleotides tested in the assayswere able to produce cytokine secretion at approximately equivalent orbetter levels than positive control oligonucleotides having a completelyphosphorothioate backbone. Negative control caused the production ofsignificantly less cytokines.

Example 2

Levels of CD69 expression (MFI) on NK cells in response to treatmentwith the test oligonucleotides versus control oligonucleotides wasexamined. CD69 expression is an indicator of T cell and NK cellactivation. The cells were exposed to the test oligonucleotides depictedin FIG. 6 by a ▴ versus a positive control oligonucleotide depicted by a▪. The test oligonucleotides shown in FIG. 6A include SEQ ID NO: 322,SEQ ID NO: 323, and SEQ ID NO: 324. The test oligonucleotides shown inFIG. 6B include SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, and SEQID NO: 328. The positive control oligonucleotide used in these studiesis SEQ ID NO: 329. Below the graphs the level of CD69 expression on Tand NK cells treated with a negative control (medium) and with LPS islisted for each experiment.

As demonstrated in FIG. 6 the oligonucleotides tested in the assays wereable to induce CD69 expression at approximately equivalent or betterlevels than positive control oligonucleotides having a completelyphosphorothioate backbone. The negative control caused the production ofsignificantly less CD69.

Example 3

Levels of interferon-alpha (IFN-α) and IL-10 produced by human PBMCfollowing exposure of these cells to the CpG oligonucleotides describedherein is shown in the attached FIGS. 7-12 and 17. The testoligonucleotides examined are depicted in the figures by a ▪. Anoligonucleotide that served as a positive control oligonucleotide SEQ IDNO: 242 was depicted by a ●. An oligonucleotide that served as anegative control oligonucleotide was depicted by a ♦ SEQ ID NO: 330. Thetest oligonucleotide shown in FIGS. 7A and 7B is SEQ ID NO: 313. Thetest oligonucleotide shown in FIGS. 8A and 8B is SEQ ID NO: 314. Thetest oligonucleotide shown in FIGS. 9A and 9B is SEQ ID NO: 319. Thetest oligonucleotide shown in FIGS. 10A and 10B is SEQ ID NO: 316. Thetest oligonucleotide shown in FIGS. 11A and 11B is SEQ ID NO: 317. Thetest oligonucleotide shown in FIGS. 12A and 12B is SEQ ID NO: 320. Thetest oligonucleotide shown in FIGS. 17A and 17B is SEQ ID NO: 321. Theconcentration of oligonucleotide used to produce a particular data pointis depicted along the X-axis (μM).

As demonstrated in FIGS. 7-12 and 17 each of the oligonucleotides testedin the assays were able to produce different levels and patterns ofcytokine secretion. For instance, at approximately equivalent or lowerconcentrations most of the tested ODN resulted in better induction ofone or more cytokines than the positive control oligonucleotide having acompletely phosphorothioate backbone. The negative control caused theproduction of significantly less cytokines.

Upon incubation with SEQ ID NO: 313 PBMC secrete similar levels ofInterferon-alpha (IFNα) and Interleukin-10 (IL-10) as after incubationwith SEQ ID NO: 242. SEQ ID NO: 314 has similar effects on the amount ofIL-10 secreted from human PBMC, as SEQ ID NO: 242, while the secretionof IFNα is strongly increased. In contrast to SEQ ID NO: 242, SEQ ID NO:319 induces only low levels of IFNα secretion from human PBMC, while theamount of secreted IL-10 is comparable between the two oligonucleotides.SEQ ID NO: 316 was able to induce several times higher levels of IFNαfrom human PBMC than SEQ ID NO: 242. An increase in the total amount ofsecreted IL-10 was also observed. SEQ ID NO: 317 demonstrated similarproperties to SEQ ID NO: 316, with strongly increased IFNα secretionfrom human PBMC compared to SEQ ID NO: 242. The levels of IL-10secretion were slightly elevated. Although SEQ ID NO: 320 resulted ininduction of IFNα and IL-10 from human PBMC the induction was less thanthat of SEQ ID NO: 242. SEQ ID NO: 321 is capable of inducing more thanten times higher levels of IFNα from human PBMC than SEQ ID NO: 242(FIG. 17A). Compared to SEQ ID NO: 242, the IL-10 secretion from humanPBMC induced by SEQ ID NO: 321 is slightly increased at higherconcentrations of this oligonucleotide (FIG. 17B).

Example 4

Levels of B cell and monocyte activation following exposure of thesecells to the CpG oligonucleotides described herein is shown in theattached FIGS. 13-15, 16 and 18-20. The test oligonucleotides examinedare depicted in the Figures by a ▪. An oligonucleotide that served as apositive control oligonucleotide SEQ ID NO: 242 was depicted by a ●. Anoligonucleotide that served as a negative control oligonucleotide wasdepicted by a ♦ SEQ ID NO: 330. The test oligonucleotide shown in FIGS.13A and 13B is SEQ ID NO: 313. The test oligonucleotide shown in FIGS.14A and 14B is SEQ ID NO: 314. The test oligonucleotide shown in FIGS.15A and 15B is SEQ ID NO: 319. The test oligonucleotide shown in FIGS.16A and 16B is SEQ ID NO: 316. The test oligonucleotide shown in FIGS.18A and 18B is SEQ ID NO: 321. The test oligonucleotide shown in FIGS.19A and 19B is SEQ ID NO: 317. The test oligonucleotide shown in FIGS.20A and 20B is SEQ ID NO: 320. The concentration of oligonucleotide usedto produce a particular data point is depicted along the X-axis (μM).

As demonstrated in FIGS. 13-15, 16 and 18-20 each of theoligonucleotides tested in the assays were able to produce differentlevels and patterns of cell surface marker expression. For instance, atapproximately equivalent or lower concentrations most of the tested ODNresulted in better induction of the cell surface markers than thepositive control oligonucleotide having a completely phosphorothioatebackbone.

The level of CD86 expression on B cells and CD80 expression on monocytesinduced by SEQ ID NO: 313 is comparable to SEQ ID NO: 242. In contrastto SEQ ID NO: 242, SEQ ID NO: 313 stimulates the cells at lowerconcentrations compared to SEQ ID NO: 242 suggesting increased potency.The levels of CD86 expression on B cells and CD80 expression onmonocytes induced by SEQ ID NO: 314 are comparable. The effects of SEQID NO: 314 are observed using a lower concentrations of SEQ ID NO: 314compared to SEQ ID NO: 242, demonstrating increased potency of SEQ IDNO: 314. On B cells, surface expression of CD86 is strongly upregulatedwith SEQ ID NO: 319, with a signal strength comparable to SEQ ID NO:242. On monocytes, only weakly elevated levels of CD80 expression can bedetected with SEQ ID NO: 319. The potency of SEQ ID NO: 319 to induceCD86 upregulation on B cells is slightly reduced compared to SEQ ID NO:242. Compared to SEQ ID NO: 242, SEQ ID NO: 316 induces higher levels ofthe activation marker CD86 on B cells (FIG. 16A), and of the activationmarker CD80 on monocytes (FIG. 16B). B cells become strongly activatedupon incubation of human PBMC with SEQ ID NO: 321 as shown by CD86expression (FIG. 18A). The level of CD86 is higher than that induced bySEQ ID NO: 242. Also the activation of monocytes as determined by CD80expression is stronger with SEQ ID NO: 321 than with SEQ ID NO: 242(FIG. 18B). SEQ ID NO: 317 induces CD86 expression on B cells atcomparable levels as SEQ ID NO: 242 (FIG. 19A), while expression of theactivation marker CD80 on monocytes is increased compared to SEQ ID NO:242 (FIG. 19B). SEQ ID NO: 320 induces CD86 expression on B cells to asimilar extent as SEQ ID NO: 2426 (FIG. 20A).

Example 5 Semi-soft Oligonucleotides are Immunostimulatory for HumanPBMC in vitro

In this example semi-soft oligonucleotides were assessed for theirability to induce cytokines and chemokines in vitro. Peripheral bloodmononuclear cells (PBMC) were obtained from three healthy human donorsand cultured in the presence of various concentrations (0.05, 0.1, 0.2,0.5, 1.0, and 5.0 μM) of fully stabilized CpG SEQ ID NO: 242 orsemi-soft SEQ ID NO:241. After 6, 16, or 48 hours, culture supernatantswere collected and various cytokines (IFN-α, TNF-α, IL-10) and thechemokine IP-10 in the supernatants were measured by ELISA. At lowconcentration of oligonucleotide, the semi-soft and fully stabilizedoligonucleotides induced IFN-α to a similar extent after 16 or 48 hoursin culture. However, maximum induction of IFN-α with ODN 5476 wasreached at about half the oligonucleotide concentration needed for SEQID NO: 242. At intermediate concentrations, SEQ ID NO: 242 induced moreIFN-α than SEQ ID NO: 241, and at high concentrations, neither SEQ IDNO: 242 nor SEQ ID NO: 241 induced much IFN-α. The chemokine IP-10 wasstimulated to a similar extent and with a similar concentrationdependence by the semi-soft and fully stabilized oligonucleotides. Inboth cases, ca. 700 pg/mL of IP-10 was observed at lower concentrationsof oligonucleotide, and less IP-10 was induced at higher concentrationsof oligonucleotide. A similar pattern to that of IP-10 was observed forthe cytokine IL-10, except that the semi-soft oligonucleotide at 0.05 μMinduced a significant amount of IL-10, whereas the fully stabilizedoligonucleotide at 0.05 μM induced little to no IL-10. Semi-soft andfully stabilized oligonucleotides were similar in their ability toinduce TNF-α, i.e., both types of oligonucleotide strongly inducedTNF-α, particularly at high concentration.

TABLE 1 Cytokines and chemokine (pg/mL)¹ induced by oligonucleotides(μM) ODN 0.05 0.1 0.2 0.5 1.0 5.0 IFN-α SEQ 534.8 (3.5) 466.0 (7.5)251.6 (22.9) 25.4 (21.4) 22.9 (26.3) 26.7 (22.1) ID NO: 241 SEQ 444.0(23.9) 573.6 (41.7) 892.4 (58.0) 583.6 (51.5) 115.6 (2.5) 51.5 (12.8) IDNO: 242 IP-10 SEQ 5677.8 (18.9) 6221.5 (22.4) 4936.6 (11.8) 1493.6 (5.5)121.9 (0.4) 0.0 (0.0) ID NO: 241 SEQ 7287.4 (5.5) 6685.8 (12.8) 6967.4(15.9) 4422.7 (11.0) 361.7 (2.6) 0.0 (0.0) ID NO: 242 IL-10 SEQ 447.6(3.7) 385.3. (4.9) 257.3 (3.1) 92.9 (1.6) 46.5 (0.2) 17.3 (1.5) ID NO:241 SEQ 73.4 (1.0) 399.8 (3.0) 367.7 (9.8) 237.8 (2.6) 52.3 (1.3) 10.5(0.3) ID NO: 242 TNF-α SEQ 179.0 (18.3) 186.4 (15.9) 229.9 (23.4) 178.8(9.0) 368.2 (22.3) 886.3 (31.7) ID NO: 241 SEQ 196.8 (25.9) 211.5 (8.7)242.7 (5.5) 262.1 (6.3) 479.8 (33.5) 939.6 (69.7) ID NO: 242 ¹Valuesreported as mean (standard deviation).

Example 6 Stimulation of Murine Macrophages in vitro by Semi-soft SEQ IDNO: 241 v. Fully Stabilized ODN

A murine macrophage cell line (RAW264) was incubated for 16 hours withsemi-soft oligonucleotide SEQ ID NO: 241, fully stabilizedoligonucleotide SEQ ID NO: 242, fully stabilized ODN 1826,lipopolysaccharide (LPS) or PBS. Semi-soft and fully stabilized ODN wereexamined at concentrations of 0.02, 0.05, and 0.1 μM. Supernatants werecollected and the p40 subunit of IL-12 (IL-12p40, pg/mL) measured byELISA. Results are shown in Table 2. Semi-soft oligonucleotide SEQ IDNO: 241 was significantly more potent at inducing macrophages to secreteIL-12p40 than either fully stabilized ODN.

TABLE 2 IL-12p40 secretion by murine macrophages stimulated by semi-softoligonucleotide SEQ ID NO: 241 ODN concentration IL-12 p40, pg/mL ODN(μM) mean (S.D.) SEQ ID NO: 241 0.02 148.8 (37.5) 0.05 149.8 (28.7) 0.1162.3 (8.4) SEQ ID NO: 242 0.02  41.4 (18.6) 0.05  42.0 (26.2) 0.1  23.0(10.7) SEQ ID NO: 386 0.02  43.5 (23.0) 0.05  38.3 (19.2) 0.1  54.4(4.1) LPS — 346.5 (20.5) PBS —  32.0 (12.1)

Example 7 Semi-soft B Class Oligonucleotides with Sequence Optimized forStimulation of Human Immune Cells are Potent Immunostimulators of MurineImmune Cells

It has been reported that human and murine immune cells respond todifferent CpG ODN. Fully stabilized CpG SEQ ID NO: 242 has beenconsidered “optimal” for stimulating human immune cells, but has notbeen considered “optimal” for stimulating murine immune cells.Conversely, fully stabilized CpG ODN 5890 (5′ T*C*A*A*C*G*T*T 3′) hasbeen considered “optimal” for stimulating murine immune cells, but hasnot been considered “optimal” for stimulating human immune cells. Bothhuman and murine B cells are reported to express TLR9. TLR9-expressingHEK293 murine splenocytes were cultured in the presence of variousconcentrations of fully stabilized CpG SEQ ID NO:242, fully stabilizedCpG ODN 5890, or semi-soft SEQ ID NO:241, and TLR9 activation wasmeasured as follows. Cells used for this assay were expressed murineTLR9 and contained a reporter gene construct. Cells were incubated withODNs for 16 h at 37° C. in a humidified incubator. Each data point wasperformed in triplicate. Cells were lysed and assayed for reporter geneactivity. Stimulation indices were calculated in reference to reportergene activity of medium without addition of ODN. Semi-softoligonucleotide SEQ ID NO: 241 and fully stabilized oligonucleotide SEQID NO: 242 have the identical base sequence. Results are shown in Table3. At the lowest concentrations, SEQ ID NO: 241 and SEQ ID NO: 242 hadminimal immunostimulatory effect. However, as concentration increased to14 nM and above, SEQ ID NO: 241 was clearly more immunostimulatory thanSEQ ID NO: 242. At the highest concentration studied in this experiment,SEQ ID NO: 241 was at least as stimulatory as the murine-optimized fullystabilized oligonucleotide ODN 5890.

TABLE 3 Stimulation index of murine TLR9 expressing HEK293 cells bysemi-soft ODN with sequence optimized for human cells ODN SEQ ID SEQ IDConc. 5890 NO: 241 NO: 242  0.9 nM 1.4 0.7 0.9  3.5 nM 2.4 1.1 1.2   14nM 12.5 1.9 1.1   58 nM 21.4 4.3 2.0 0.23 μM 25.2 12.0 6.2 0.94 μM 28.618.3 8.0 3.75 μM 29.3 32.1 10.3

Example 8-9 Semi-soft Oligonucleotides Induce NK Cell Activation

Semi-soft and fully stabilized oligonucleotides were also compared interms of their ability to stimulate NK cell activation. Using a standardchromium release assay, 10×10⁶ BALB/c spleen cells were cultured in 2 mLRPMI supplemented with 10% FBS (heat inactivated to 65° C. for 30 min.),50 μM 2-mercaptoethanol, 100 U/mL penicillin, 100 μg/mL streptomycin,and 2 mM L-glutamate, with or without either semi-soft SEQ ID NO: 241 orfully stabilized SEQ ID NO: 242, each ODN added to a final concentrationof 1, 3, or 10 μg/mL, for 48 hours. Cells were washed and then used aseffector cells in a short-term ⁵¹Cr release assay with YAC-1 and 2C11,two NK-sensitive target cell lines (Ballas Z K et al. (1993) J Immunol150:17-30). Effector cells were added at various concentrations to 10⁴⁵¹Cr-labeled target cells in V-bottom microtiter plates in 0.2 mL, andincubated in 5% CO₂ for 4 hr at 37° C. Effector cell:target cell (E:T)ratios studied were 6.25:1, 25:1, 50:1, and 100:1. Plates were thencentrifuged and an aliquot of the supernatant counted for radioactivity.Percent specific lysis was determined by calculating the ratio of the⁵¹Cr released in the presence of effector cells minus the ⁵¹Cr releasedwhen the target cells are cultured alone, over the total counts releasedafter cell lysis in 2% acetic acid (100 percent lysis) minus the ⁵¹Crcpm released when the cells are cultured alone. Results are shown inTable 5 below. In summary, semi-soft oligonucleotide SEQ ID NO: 241 andfully stabilized SEQ ID NO: 242 induced essentially comparable levels ofNK cell activation over all ODN concentrations and E:T ratios examined.

TABLE 5 NK cell-mediated specific lysis E:T Ratio ODN μg/mL 6.25:1 25:150:1 100:1 SEQ ID 1 8 17 17.5 27.5 NO: 241 3 2.5 5 8 15 10 4 12.5 20 28SEQ ID 1 7 8 12.5 22 NO: 242 3 3.5 4 11 18 10 5 12.5 23 32.5

Example 10 Semi-soft Oligonucleotides are Generally moreImmunostimulatory than All-phosphorothioate Oligonucleotides of the Sameor Similar Sequence

All tested semi-soft versions were more active in the human TLR9 assaythan the corresponding uniformly phosphorothioate molecule (Table 6).The average stimulation index was calculated from data points of fourconcentrations (0.1 μM, 0.5 μM, 2 μM, and 8 μM). In the Table, Urepresents 2′-deoxyuracil.

TABLE 6 Relative average stimulation indices ofsemi-soft oligonucleotides versus all-phosphorothioate oligonucleotides of the same or similar sequenceRelative Average Stimulation Sequence IndexT*C*G*T*C*G*T*T*T*T*C*G*G*C*G*G*C*C*G*C*C*G (SEQ ID NO: 247) 1.00T*C*G*C*C*G*T*T*T*T*C_G*G*C_G*G*C*C_G*C*C*G (SEQ ID NO: 248) 0.74T*C*G*C*C*G*T*T*T*T*C_G*G*C_G*G*C*C_G*C*C*G (SEQ ID NO: 249) 0.72T*C*G*T*C*G*T*T*T*T*C_G*G*C_G*G*C*C_G*C*C*G (SEQ ID NO: 250) 1.37T*C*G*T*C*G*T*T*T*T*C_G*G*C_G*G*C*C_G*C*C*G (SEQ ID NO: 251) 1.25T*C_G*C*C_G*T*T*T*T*C_G*G*C_G*G*C*C_G*C*C*G (SEQ ID NO: 252) 2.99T*C_G*C*C_G*T*T*T*T*C_G*G*C_G*G*C*C_G*C*C*G (SEQ ID NO: 253) 2.22T*C_G*T*C_G*T*T*T*T*C*G*G*C*G*G*C*C*G*C*C*G (SEQ ID NO: 254) 3.46T*C_G*T*C_G*T*T*T*T*C*G*G*C*G*C*C*C*G*C*C*G (SEQ ID NO: 255) 4.08T*C_G*T*C_G*T*T*T*T*C_G*G*C_G*G*C*C_G*C*C*G (SEQ ID NO: 256) 5.69T*C_G*T*C_G*T*T*T*T*C_G*G*C_G*G*C*C_G*C*C*G (SEQ ID NO: 257) 4.49T*G*T*C*G*T*T*G*T*C*G*T*T*G*T*C*G*T*T*G*T*C*G*T*T (SEQ ID NO: 244) 1.00T*G*T*C_G*T*T*G*T*C_G*T*T*G*T*C_G*T*T*G*T*C_G*T*T (SEQ ID NO: 258) 4.23T*G*T*C_G*T*T*G*T*C_G*T*T_G*T*C_G*T*T_G*T*C_G*T*T (SEQ ID NO: 243) 4.74T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T (SEQ ID NO: 259) 1.00T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T*T*T*G*T*C_G*T*T (SEQ ID NO: 260) 1.80T*C*G*T*C*G*T*T*T*T*G*A*C*G*T*T*T*T*G*T*C*G*T*T (SEQ ID NO: 261) 1.00T*C_G*T*C_G*T*T*T*C_G*A*C_G*T*T*T*T*G*T*C_G*T*T (SEQ ID NO: 262) 2.71T*C_G*T*C_G*T*T*T*T_G*A*C_G*T*T*T*T*G*T*C*G*T*T (SEQ ID NO: 263) 3.01T*C_G*T*C_*T*T*T*T_G*A*C_G*T*T*T*T*G*T*C_G*T*T (SEQ ID NO: 264) 3.06T*C_G*T*C_G*T*T*T*T_G*A*C_G*T*T*T*T (SEQ ID NO: 265) 2.06T*C_G*T*C_G*T*T*T*T_G*A*C_G*T*T (SEQ ID NO: 266) 1.43T*C_G*T*C_G*T*T*T*C_G*A*C*G*T*T (SEQ ID NO: 267) 0.91G*T*T*C*T*C*G*C*T*G*G*T*G*A*G*T*T*T*C*A (SEQ ID NO: 268) 1.00G*T*T*C*T*C_G*C*T_G*G*T_G*A*G*T*T*T*C*A (SEQ ID NO: 269) 3.45T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T (SEQ ID NO: 270) 1.00T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T (SEQ ID NO: 271) 2.49T*C_G*T*C_G*T*T*T*U_G*T*C_G*T*T*T*T_G*T*C_G*T*T (SEQ ID NO: 272) 2.51T*C*G*T*C*G*T*T*T*U*G*T*C*G*T*T*T*T*G*T*C*G*T*T (SEQ ID NO: 273) 1.00T*C_G*T*C_G*T*T*T*U_G*T*C_G*T*T*T*T_G*T*C_G*T*T (SEQ ID NO: 274) 2.62T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T (SEQ ID NO: 242) 1.00T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T*T*T_G*T*C_G*T*T (SEQ ID NO: 276) 1.95T*C*G*U*C*G*T*T*T*T*G*T*C*G*T*T*T*U*G*U*C*G*T*T (SEQ ID NO: 277) 1.00T*C_G*U*C_G*T*T*T*T_G*T*C_G*T*T*T*U_G*U*C_G*T*T (SEQ ID NO: 278) 1.39T*C*G*T*C*G*U*U*U*T*G*T*C*G*U*U*U*U*G*T*C*G*T*T (SEQ ID NO: 279) 1.00T*C_G*T*C_G*U*U*U*C_G*T*C_G*U*U*U*U_G*T*C_G*T*T (SEQ ID NO: 280) 2.05A*A*C*G*T*C*G*T*T*T*T*C*G*T*C*G*T*T (SEQ ID NO: 281) 1.00A*A*C_G*T*C_G*T*T*T*T*C_G*T*C_G*T*T (SEQ ID NO: 282) 1.58

Example 11 Improved in vitro Potency of Semi-soft Versions of WeaklyImmunostimulatory Fully Stabilized Oligonucleotides

(T*G*T*C*G*T*T*G*T*C*G*T*T*G*T*C*G*T*T*G*T*C*G*T*T, SEQ ID NO:244) is afully stabilized, all-phosphorothioate CpG oligonucleotide with lowimmunostimulatory potency compared to SEQ ID NO: 242. Related semi-softoligonucleotides (T*G*T*C_G*T*T*G*T*C_G*T*T*G*T*C_G*T*T*G*T*C_G*T*T, SEQID NO:258) and (T*G*T*C_G*T*T*G*T*C_G*T*T_G*T*C_G*T*T_G*T*C_G*T*T, SEQID NO:243) were many-fold more potent than SEQ ID NO: 244 and even morepotent than SEQ ID NO: 242.

T*G*T*C_G*T*T*G*T*C_G*T*T*G*T*C_G*T*T*G*T*C_G*T*T (SEQ ID NO: 258)T*G*T*C_G*T*T*G*T*C_G*T*T_G*T*C_G*T*T_G*T*C_G*T*T (SEQ ID NO: 243)T*G*T*C*G*T*T*G*T*C*G*T*T*G*T*C*G*T*T*G*T*C*G*T*T (SEQ ID NO: 244)

TABLE 7 Improved immune stimulation by semi-soft variants of a fullystabilized but weakly immunostimulatory oligonucleotide ODNconcentration, μM ODN 0.1 0.5 2 8 SEQ ID 16.0 47.5 71.4 68.5 NO: 244 SEQID 19.3 40.5 78.2 77.9 NO: 243 SEQ ID 2.6 9.5 12.9 14.0 NO: 241 SEQ ID10.6 34.2 38.3 40.8 NO: 242

Example 12 Semi-soft Oligonucleotides of Reduced Length areImmunostimulatory in vitro

Semi-soft 16-mer, SEQ ID NO:283, 16-mer, SEQ ID NO:245, 17-mer, SEQ IDNO:284, and 24-mer, SEQ ID NO:241 were compared with fully stabilizedODNs 24-mer, SEQ ID NO:242 and 18-mer, SEQ ID NO:285 in terms of theirability to stimulate TLR9 signaling. Each oligonucleotide was added toHEK293 cells transfected with human TLR9 and a reporter gene constructat a concentration of 1, 6, 12, or 24 μg/mL, and TLR9 activation wasmeasured as described above.

(16-mer) T*C_G*T*C_G*T*T*T*T*C_G*T*C_G*T (SEQ ID NO: 283) (16-mer)T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T (SEQ ID NO: 245) (17-mer)T*C_G*T*C_G*T*T*T*T*C_G*T*C_G*T*T (SEQ ID NO: 284) (18-mer)A*A*C*G*T*C*G*T*T*T*T*C*G*T*C*G*T*T (SEQ ID NO: 285) (24-mer)T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T*T*T*G*T*C_G*T*T (SEQ ID NO: 241)(24-mer) T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T(SEQ ID NO: 242)

While the 18-mer fully stabilized oligonucleotide ODN SEQ ID NO: 285 wasless immunostimulatory than the 24-mer fully stabilized oligonucleotideSEQ ID NO: 242 at all concentrations examined, the 16-mer and 17-mersemi-soft oligonucleotides were at least as stimulatory as 24-mer SEQ IDNO: 242 at concentrations of 6 μg/mL and above. In addition, the 16-merand 17-mer semi-soft oligonucleotides were nearly as immunostimulatoryas 24-mer semi-soft oligonucleotide SEQ ID NO: 241.

TABLE 8 Immunostimulatory activity of short semi-soft oligonucleotidescompared with short and long fully stabilized and semi-softoligonucleotides ODN concentration, μg/mL ODN 1 6 12 24 SEQ ID 1.2 17.129.0 39.5 NO: 283 SEQ ID 1.1 8.4 31.3 48.9 NO: 245 SEQ ID 3.4 23.9 35.945.6 NO: 284 SEQ ID 4.6 12.9 15.9 18.0 NO: 285 SEQ ID 6.4 33.0 50.8 58.6NO: 241 SEQ ID 11.0 24.6 26.2 21.9 NO: 242

Example 13 Semi-soft Oligonucleotides are Immunostimulatory in vivo

BALB/c mice were divided into groups and administered subcutaneously 400μg semi-soft oligonucleotide SEQ ID NO: 241, fully stabilizedimmunostimulatory oligonucleotide SEQ ID NO: 242, fully stabilizednegative control oligonucleotide (TGCTGCTTTTGTGCTTTTGTGCTT, SEQ IDNO:286), or an equivalent volume of phosphate-buffered saline (PBS).Animals were bled 3 hours after injection and serum levels of IP-10,IFN-γ, and TNF-α determined using appropriate cytokine-specific ELISA.Serum IP-10 was about two times higher in animals receiving semi-softSEQ ID NO: 241 (8,000-12,000 pg/mL) than fully stabilizedimmunostimulatory oligonucleotide SEQ ID NO: 242 (3,500-8,000 pg/mL).Serum IP-10 in animals receiving control SEQ ID NO: 286 had the same lowlevel of IP-10 as animals receiving PBS. Semi-soft oligonucleotide SEQID NO: 241 and fully stabilized immunostimulatory oligonucleotide SEQ IDNO: 242 induced similar amounts of IFN-γ, ca. 150 pg/mL. Semi-softoligonucleotide SEQ ID NO: 241 induced 30-45 percent more TNF-α thanfully stabilized immunostimulatory oligonucleotide SEQ ID NO: 242 (ca.1,550 pg/mL versus ca. 1,175 pg/mL in one experiment and ca. 710 pg/mLversus 490 pg/mL in another experiment.

In another set of in vivo experiments, semi-soft and fully stabilizedoligonucleotides were examined for their ability to treat tumors inBALB/c mice. Three groups of BALB/c mice were injected i.p. with murinerenal adenocarcinoma of spontaneous origin (Renca) cells, using anestablished tumor model. Salup R R et al. (1985) J Immunopharmacol7:417-36. Each group of mice also received either 100 mg semi-softoligonucleotide SEQ ID NO: 241, 100 mg fully stabilizedimmunostimulatory oligonucleotide SEQ ID NO: 242, or an equivalentvolume of PBS. Mice were followed for survival and for tumor size atdeath. Mice receiving sham treatment with PBS had a median survival of44 days and 20 percent survival at 50 days. In contrast, mice receivingsemi-soft oligonucleotide SEQ ID NO: 241 had 80 percent survival at 50days, and mice receiving fully stabilized immunostimulatoryoligonucleotide SEQ ID NO: 242 had 70 percent survival at 50 days. Interms of tumor size (cubic millimeters), after 52 days mice receivingPBS had tumor volumes of nearly 1200 mm³, while mice receiving semi-softoligonucleotide SEQ ID NO: 241 or fully stabilized immunostimulatoryoligonucleotide SEQ ID NO: 242 had tumors of ca. 250 mm³ and 180 mm³,respectively. Thus the semi-soft oligonucleotide and the fullystabilized oligonucleotide were both highly effective in reducing tumorburden and extending survival in this model experiment.

Example 14 Soft or Semi-soft Oligonucleotides have ReducedNephrotoxicity

It has been observed that administration of fully stabilizedimmunostimulatory oligonucleotides to monkeys can be associated withdevelopment of glomerulonephritis, i.e., kidney inflammation.Glomerulonephritis can be diagnosed and monitored by the presence of redblood cells and protein in the urine, often accompanied by reducedglomerular filtration rate (with azotemia), water and salt retention,hypertension, and edema. Normally urine is essentially free of bloodcells and plasma proteins. Diagnosis can also be made by renal tissuehistologic examination. Kidney tissue is reported to be rich innucleases, which are expected to be more active on soft oligonucleotidesthan on fully stabilized immunostimulatory oligonucleotides.

Monkeys are divided into two groups, one administered softoligonucleotides, and the other administered fully stabilizedimmunostimulatory oligonucleotides. The soft oligonucleotides and thefully stabilized immunostimulatory oligonucleotides are identical insequence and differ in their internucleotide linkages only. Both groupsof monkeys receive the same dose of immunostimulatory oligonucleotide.Pretreatment (baseline) and periodic on-treatment measurements are madeof at least one parameter useful for assessing for the presence ofglomerulonephritis, including, for example, dipstick urinalysis for thepresence of proteinuria and/or hematuria, microscopic urine analysis forthe presence of red blood cells and/or red blood cell casts, urineprotein concentration, blood urea nitrogen (BUN), serum creatinine,blood pressure, body weight, and kidney biopsy with light and/orelectron microscopic tissue analysis. Clinical findings are correlatedwith the type of immunostimulatory oligonucleotide administered to eachmonkey, and results are compared between groups for statisticalsignificance.

Optionally, additional paired groups of monkeys, administered eithersoft or semisoft oligonucleotides or fully stabilized immunostimulatoryoligonucleotides as above but using higher or lower oligonucleotidedose(s), are included to evaluate results further as a function ofoligonucleotide dose.

Monkeys receiving soft oligonucleotides are significantly less prone todevelop glomerulonephritis than monkeys receiving fully stabilizedimmunostimulatory oligonucleotides.

Example 15 Soft Oligonucleotides have Increased ImmunostimulatoryPotency at High Concentration

Soft oligonucleotides were compared with SEQ ID NO: 242 for theirability to induce TLR9 activity. Soft ODN and control SEQ ID NO: 242were compared at each of four concentrations, 1 μg/ml, 6 μg/ml, 12μg/ml, and 24 μg/ml. The ratios at each concentration of activation byeach soft oligonucleotide compared to activation by SEQ ID NO: 242 areshown in Table 9 below. These results indicate that softoligonucleotides are more immunostimulatory than SEQ ID NO: 242 at thehigher concentrations examined.

T*G*T*C_G_T*T*G*T*C_G_T*T*G*T*C_G_T*T*G_T*C_G*T*T SEQ ID NO: 287T*C_G_T*T*T*T*T*T*T*C_G_T*T*T*T*T*T*T*C_C_T*T*T SEQ ID NO: 288T*C_G*T*C_G*T*T*T*T*T*C_G_G*T*C_G_T*T*T*T SEQ ID NO: 289T*C_G_T*C_G_T*T*T*T*T*C_G_T*G*C_G_T*T*T*T*T SEQ ID NO: 290T*C_G_T*C_G_T*T*T*T*C_G_T*T*T*T*T*T*T*C_G*T*T*T SEQ ID NO: 291T*C_G_T*T*T*T*G*T*C_G_T*T*T*T*T*T*T*C_G*A SEQ ID NO: 292T*C_G_T*C_G_T*T*T*T_G_T*C_G_T*T*T*T_G*T_C_G*T*T SEQ ID NO: 293

TABLE 9 Relative potency of soft oligonucleotides compared to SEQ ID NO:242 at each concentration ODN concentration, μg/mL ID 1 6 12 24 SEQ IDNO: 0.11 0.12 1.00 1.68 287 SEQ ID NO: 0.30 0.62 1.67 1.81 288 SEQ IDNO: 0.13 0.52 1.67 1.97 289 SEQ ID NO: 0.18 0.41 1.69 2.27 290 SEQ IDNO: 0.16 0.35 1.56 1.81 291 SEQ ID NO: 0.25 0.48 1.38 1.84 292 SEQ IDNO: 0.10 0.11 1.20 2.05 293

Example 16 Oligonucleotide Stability in Serum and in Tissues

Mice were injected subcutaneously with 25 mg/kg of semi-softoligonucleotide SEQ ID NO: 241, soft oligonucleotide

(T*C*G*T*C*G*T*T*T*T_G_T_C_G_T*T*T*T*G*T*C*G*T*T;, SEQ ID NO: 294)or fully stabilized oligonucleotide SEQ ID NO: 242. Tissue and serumsamples were obtained after selected number of hours and analyzed forintact oligonucleotide and fragments thereof.

Tissue or serum samples were spiked with a known amount of internalstandard ODN (1.25 μg polyT) and ODN were isolated from tissue andplasma samples by solid phase extraction (SPE) methods described below.The resulting solutions containing the analyte, metabolites, andinternal standard were analysed by capillary gel electrophoresis (CGE)and MALDI-TOF methods also described below. Total amounts of therecovered ODN (i.e., analyte plus metabolites) from kidney, liver,spleen, and serum samples analysed by CGE were defined. A standarddeviation was calculated. The relative amount in percent of the totalpeak area was assigned to each metabolite.

SPE. For isolation of ODN from serum, 100 μg of the sample was spikedwith 1.25 μg internal standard ODN, mixed and dissolved in 5 mlSAX-buffer. This solution was applied on an anion exchange column (SAX,Agilent), the column was washed and ODN eluted with a buffer ofincreased ionic strength. The resulting eluate was desalted using areversed phase (RP) column (Glen Research) or a comparable column (HLB,Waters). The eluates from the RP column, containing only water andacetonitrile were dried and solubilized in the same tube in 60 μldeionized water. For further desalting of the samples a membranedialysis was performed. Samples were analysed directly by capillary gelelectrophoresis. For MALDI-TOF MS, samples were used either undiluted orconcentrated, i.e., 50 μl of the ODN sample were dried in a vacuum anddissolved in deionized water and assayed as described below.

ODN from tissues were isolated according to a similar SPE protocol. 100mg of tissue was homogenised using a FastPrep device. Proteinase K wasadded and proteins hydrolysed for 2 h. A phenol extraction was performedbefore proceeding with the water soluble fraction in the SPE methoddescribed above.

CGE. The desalted samples containing analyte, its metabolites, and adefined amount of internal standard ODN were electrokinetically injectedinto a gel-filled capillary (neutral, 30 cm, eCAP DNA capillary, Beckman# 477477) at the sample side with water pre-injection. A voltage of 300V/cm was applied while detection was monitored at 260 nm. Separation wascarried out at 25° C. in Tris/boric acid/EDTA buffer containing 7M urea.The analyte was identified by its relative migration time(MT_(Oligo)/MT_(Int Std.)) compared to that of a standard which issimilarly prepared and concomitantly analysed. The relative migrationtime and relative area percent of any electrophoretic peak that is >3×signal:noise (S:N) ratio was recorded. Peak heights of between 3× and10× signal:noise were recorded as not quantifiable.% Oligo=(peak area/total peak area>3×S:N)×100%

MALDI-TOF. The desalted samples containing the analyte and itsmetabolites were analysed on an Applied Biosystems MALDI-TOF massspectrometer with a delayed extraction source, a nitrogen laser at 337nm wavelength, and a 1.2 meter flight tube. Instrument settings were asfollows: voltage 25 kV; grid voltage 95.4%; guide wire 0.1%; delay time1200 nsec. As matrix 3-hydroxypicolinic acid containing diammoniumcitrate was used. The spectra of the ODN samples were calibratedexternally on the same plate under identical conditions using a set ofstandard ODN of known molecular weights.

Results obtained at 48 hours are shown in FIG. 20. FIG. 20 shows that inthe kidney semi-soft SEQ ID NO: 241 and soft ODN SEQ ID NO: 294 werereduced dramatically (by 93 percent and by 87 percent, respectively)compared with all-phophorothioate SEQ ID NO: 242.

Example 17 C Oligonucleotides are Immunostimulatory in vitro

Semi-soft C-Class oligonucleotides were prepared with phophodiesterlinkages within the 5′ non-palindromic portion (ODN SEQ ID NO: 255), the3′ palindromic portion (ODN SEQ ID NO: 251), and within both the 5′non-palindromic portion and the 3′ palindromic portion (ODN SEQ ID NO:295). In addition, ODN SEQ ID NO: 252 was prepared with linkages likeODN SEQ ID NO: 295 but with 2′-O-Me ribose sugars in the nucleotidesmaking up the 3′ palindromic portion (shown underlined below). Theseoligonucleotides were then evaluated using a TLR9 assay described above.

T*C_G*T*C_G*T*T*T*T*C*G*G*C*G*G*C*C*G*C*C*G (SEQ ID NO: 255)T*C*G*T*C*G*T*T*T*T*C_G*G*C_G*G*C*C_G*C*C*G (SEQ ID NO: 251)T*C_G*T*C_G*T*T*T*T*C_G*G*C_G*G*C*C_G*C*C*G (SEQ ID NO: 295)T*C_G*C*C_G*T*T*T*T*C_G*G*C_G*G*C*C_G*C*C*G (SEQ ID NO: 252)

C-Class oligonucleotides with fully stabilized backbones generallyexhibit relatively low TLR9 activity compared with B-Classoligonucleotides. As shown in Table 10 below, incorporation of semi-softsequence in just the 5′ non-palindromic portion (ODN SEQ ID NO: 255)significantly enhanced TLR9 activity compared to incorporation ofsemi-soft sequence in just the 3′ palindromic portion (ODN SEQ ID NO:251). Incorporation of semi-soft sequence in both the 5′ non-palindromicportion and the 3′ palindromic portion (ODN SEQ ID NO: 295) resulted inenhanced TLR9 activity compared to incorporation of semi-soft sequencein just the 3′ palindromic portion (ODN SEQ ID NO: 251).

TABLE 10 TLR9 stimulation by semi-soft C-Class oligonucleotides ODNconcentration, μg/mL ODN 0.1 0.5 2.0 8.0 SEQ ID 2.3 16.9 36.4 35.7 NO:255 SEQ ID 1.2 2.5 8.4 16.8 NO: 251 SEQ ID 2.0 11.6 29.8 37.3 NO: 295SEQ ID 1.1 3.9 22.1 47.0 NO: 252

Semi-soft C-Class oligonucleotides not only retain their ability toinduce IFN-α by human PBMC, but they also are significantly more potentat low concentrations. The enhanced potency was most pronounced in thoseC-Class oligonucleotides that included semi-soft sequence in the 5′non-palindromic portion (ODN SEQ ID NO: 255 and ODN SEQ ID NO: 295). ODNSEQ ID NO: 255, SEQ ID NO: 251, and SEQ ID NO: 295 were evaluated byELISA and compared with SEQ ID NO: 242, the fully stabilized form ofthese three semi-soft oligonucleotides and a potent C-Classoligonucleotide inducer of IFN-α. Results are presented in Table 11.

TABLE 11 IFN-α induction (pg/mL) by semi-soft versions of C-Classoligonucleotide ODN concentration, μM ODN 0.1 0.2 0.5 1.0 2.0 SEQ ID3202 7429 937 64 3 NO: 255 SEQ ID 688 3033 3083 NO: 251 SEQ ID 2560 33633246 930 41 NO: 295 — 50 504 3247 2114 1789

Example 18 Physicochemical Characteristics of SEQ ID NO. 313

Methods:

The powder X-ray diffractometric pattern of SEQ ID NO. 313 showed a halowhich is characteristic of an amorphousphase. Water vapor sorptionanalysis has shown SEQ ID NO. 313 to be highly hygroscopic. The tendencyof the drug to exchange moisture may result in varying amount ofmoisture depending on the humidity of the environment. The compoundexhibits high water solubility (>100 mg/mL) and thus has adequatesolubility throughout the useable pH range. Analysis of aqueoussolutions of the drug at elevated temperature show that it degradesrapidly in mildly acidic to acidic environments, but solutions bufferedabove pH six appear to have adequate solution stability.

Results:

SEQ ID NO. 313 was found to be amorphous in nature and highlyhygroscopic. The compound exhibits high water solubility (>100 mg/mL)and thus has adequate solubility throughout the useable pH range. TheODN degrades rapidly in mildly acidic to acidic environments. Solutionsbuffered above pH six appear to have adequate solution stability.

Example 19 Stimulation of TLR9-Transfected Cells in vitro

Methods:

HEK 293 cells transfected with human TLR9 were incubated with SEQ. IDNo. 313 or SEQ ID No. 329 for 16 hours. The signal was determined by aluciferase readout.

Results

Compared with SEQ ID No. 329, SEQ ID No. 313 was a more potentstimulator of the target receptor TLR9.

Example 20 Stimulation of Human Immune Cells in vitro

Methods:

Human peripheral blood mononuclear cells from 6 donors were incubatedwith SEQ ID No. 313 or SEQ ID No. 329 for 24 or 48 hours. Secretion ofcytokines were measured.

Results

The results are shown in FIG. 23. Compared with SEQ ID No. 329, SEQ IDNo. 313 showed increased or at least simillar efficacy and/or potency asan inducer of TLR9-associated cytokines IL-6, IL-10, IFNα and IP-10.

Example 21 Stimulation of Murine Splenocytes in vitro

Methods:

Murine (BALB/c) splenocytes were incubated with SEQ ID No. 313 or SEQ IDNo. 329 for 48 hours. Secretion of cytokines and IP-10 were measured.

Results

Compared with SEQ ID No. 329, SEQ ID No. 313 showed increased or atleast simillar efficacy and/or potency as an inducer of cytokines IL-6,IL-10, IL-12p40, IFNα, TNFα and IP-10. The data is shown in FIG. 24.This data demonstrates that the activity of SEQ ID No. 313 on murineimmune cells is comparable to that on human cells (above) and issimilarly consistent with activation via TLR9.

Example 22 Cytokine Gene Induction in Mice in vivo

Methods:

This study assessed expression of cytokines in mouse lungs after SEQ IDNo. 313 was dosed into the airways. To investigate kidney exposure,induction of the same cytokines (as described in Examples 10 and 21) inthis organ was also assessed. Mice (male, BALB/c) were dosed with SEQ IDNo. 313 or SEQ ID No. 329 (each 1 mg/kg) either by intranasalinstillation or by bolus intravenous injection. Lungs and kidney wereremoved 8 or 15 hours after dosing. RNA was extracted and reversetranscribed to cDNA. Target fragments of cDNA were amplified anddetected by real-time PCR (Roche LightCycler using SYBR Green detectionmethod). The primers for GAPDH, IFN gamma, IL-6, IP-10, and TNF-alphawere designed using the LC PROBE DESIGN software from Roche (Version 1.0Roche catalog No 3 139 174. The primers for IFNalpha were designed usingPRIMER 3 software. Product yield was normalized as the ratio of controlgene (GAPDH) expression.

Results

When dosed into the airways, SEQ ID No. 313 induced expression ofTLR9-associated genes (IL-6, TNFα, IFNα, IFNγ and IP-10) in the lung.The results are shown in FIG. 25. However, with the exception of IP-10,these genes were not expressed in kidneys of mice dosed by this route.Since IP-10 is typically induced by interferons, is expression of thischemokine could have occurred indirectly as a result of interferonssecreted into the systemic circulation from the lung. When SEQ ID No.313 was administered intravenously, each of these genes except IFNγ wasinduced in kidney. Therefore, the lack of renal impact of the SEQ ID No.313 after dosing into the airways was likely due to low systemicexposure.

CpG ODNs may cause renal effects through a number of mechanisms. Anacute renal granulomatous inflammation caused by a TLR9-dependentmechanism has been observed after systemic exposure to some CpG ODNs.Our results suggest that systemic exposure to SEQ ID No. 313administered into the airways is not sufficient to directly induceTLR9-associated genes in the kidney.

Example 23 Effects on Antigen-induced Lymph Node Development in Mice invivo

Methods:

This study investigated the ability of SEQ ID No. 313 to induce immunedeviation away from a Th2-type response in draining lymph nodes of mice.Mice (male, BALB/c) were sensitized by injection into the right rearfootpad with antigen (ovalbumin, 100 μg) in complete Freund's adjuvant.Mice were simultaneously injected into the same footpad with SEQ ID No.313 or SEQ ID No. 329 (1.5 mg/kg) or vehicle (saline). Six days afterfootpad injection, the draining popliteal lymph node was removed. Tcells (CD3⁺) and B cells (B220⁺) were counted by flow cytometry. An exvivo antigen recall assay was performed as follows 1×10⁶ cells (from thedraining popliteal lymph node) were incubated in 220 ul medium RPMI1640+10% fetal bovine serum containing either ovalbumin (100 ug/ml) ordiluent. After 36 hoursculture medium was removed and the concentrationsof IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, GM-CSF, IFNgamma, and TNF alpha were measured using a kit from LINCO research, Inc.14 research Park Drive, St Charles, Mo. 63304 and analysed on theLuminex multiplex system (Luminex Corporation, 12212 TechnologyBoulevard, Austin, Tex. 78727-6115).

Results

Cell numbers in popliteal lymph nodes Sensitization caused accumulationsof T cells and B cells in draining popliteal lymph nodes. Theseantigen-induced accumulations were not significantly increased furtherin mice that also received a CpG ODN. However, each CpG ODN injectedalone to unsensitized mice did cause both T cell and B cellaccumulations. The data is shown in FIG. 26.

Antigen recall assay Draining lymph node cells taken fromantigen-sensitized mice secreted IL-4, IL-5, IL-10 and IFNγ whenrestimulated with antigen ex vivo. In sensitized mice that also receiveda CpG ODN, secretions of Th2-type cytokines IL-4, IL-5 and IL-10 werereduced, whereas secretion of the Th1-type cytokine IFNγ was increased.Our data, shown in FIG. 27, supports the hypothesis that SEQ ID No. 313,like SEQ ID No. 329, suppresses a Th2 response to antigen sensitization.Results are mean±s.e.m. (n=9-10). * P<0.05 compared with sensitized,vehicle-treated group (Kruskal-Wallis multiple comparison test).

Example 24 Effects on Antigen-induced IgE Production in Mice in vivo

Methods:

Mice (male BALB/c) were sensitized on study days 0 and 7 with antigen(ovalbumin, 100 μg, i.p.) with aluminum hydroxide adjuvant. Micereceived SEQ ID No. 313 (0.15 or 1.5 mg/kg, i.p.) or SEQ ID No. 17 (1.5mg/kg, i.p.) two days before each sensitization and on the day of eachsensitization. Serum was collected on study day 18. Titers of antigen(ovalbumin)-specific IgE and IgG2a were measured by ELISA. A summary ofthe protocol is shown in Table 12.

TABLE 12 Summary of study protocol Sensitize Sensitize ↓ ↓ ODN ODN ODNODN ↓ ↓ ↓ ↓ Day: −2 0 5 7 18 ↓ Endpoint

Results

In mice treated with SEQ ID No. 313 or SEQ ID No. 329, production ofantigen-specific IgE was completely prevented. In contrast, productionof IgG2a was increased. Since IgE and IgG2a production arecharacteristic of Th2-type and Th1-type responses respectively, thiseffect is further evidence that SEQ ID No. 313 can suppress Th2-typeresponses to antigen sensitization. Alternatively CpG ODNs may directlyinduce T-beta expression and class switching from IgE in B cells. Thedata are shown in FIG. 28. Results are mean±s.e.m. (n=10-12, except 5for the SEQ ID NO: 329 group). * P<0.05 compared with sensitized,vehicle-treated group (Kruskal-Wallis multiple comparison test).

Example 25 Effects Against Antigen-Induced Airways Inflammation in Micein vivo

Methods:

Mice (male BALB/c) were sensitized on study days 0 and 7 with antigen(ovalbumin, 100 μg, i.p.) with aluminum hydroxide adjuvant. Mice wereantigen challenged by exposure to inhaled ovalbumin aerosol, twice eachweek for two consecutive weeks. The first challenge was on study day 21.SEQ ID No. 313 (0.1-1000 μg/kg), SEQ ID No. 329 (1-1000 μg/kg) orvehicle (saline, 20 μl) were administered into the airways by intranasalinstillation once each week, two days before the first antigen challengeof the week. Endpoints were assessed 48 hours after the last antigenchallenge. Cells in airways were recovered by bronchoalveolar lavage anddifferential cell counts were made. Eosinophil numbers (eosinophilvolume density) and mucus secretion (PAS staining) in lung tissue weredetermined by histopathological assessment. The protocol is outlined inTable 13.

TABLE 13 Summary of study protocol Sensitize Challenge Challenge ↓ ↓ ↓ ↓↓ ↓ ODN ODN ↓ ↓ Day: 0 7 19 21 24 26 28 31 33 ↓ Endpoints

Results

Antigen challenge caused an increase in the total number of leukocytes,predominantly eosinophils, in the airway lumen. The data are shown inFIG. 29. The eosinophilia was suppressed significantly in a dose-relatedmanner by SEQ ID No. 313 or SEQ ID No. 329. ED₅₀ values againsteosinophilia were: SEQ ID No. 313: 23 μg/kg; SEQ ID No. 329: 47 μg/kg.Challenge also caused an accumulation of CD4⁺ T cells (CD3⁺CD4⁺ cells)that was significantly suppressed by SEQ ID No. 313. SEQ ID No. 313 alsosignificantly suppressed antigen-induced eosinophil accumulation in lungtissue and epithelial mucus secretion. Results in FIG. 29 aremean±s.e.m. (n=15). * P<0.05 compared with antigen challenged,vehicle-treated group (Kruskal-Wallis multiple comparison test). Resultsin FIG. 30 are mean±s.e.m. (n=6). * P<0.05, ** P<0.001 compared withantigen challenged, vehicle-treated group (ANOVA, Dunnett's multiplecomparison test).

Example 26 Effects Against Antigen-induced Airways Hyperreactivity inMice in vivo

Methods:

Mice (male BALB/c) were sensitized on study days 0 and 7 with antigen(ovalbumin, 100 μg, i.p.) with aluminum hydroxide adjuvant. Mice wereantigen challenged by exposure to inhaled ovalbumin aerosol, twice eachweek for two consecutive weeks. The first challenge was on study day 19.SEQ ID No. 313 (10-1000 μg/kg) or vehicle (saline, 20 μl) wereadministered intranasally once each week, two days before the firstantigen challenge of the week. Airways hyperreactivity was assessed 24hours after the last antigen challenge by measuring bronchoconstriction(increase in airway resistance) to intravenous methacholine. For eachanimal, a dose-response curve to methacholine was obtained, and airwayreactivity was quantified as the area under the curve. The protocol isshown in Table 14.

TABLE 14 Summary of study protocol Sensitize Challenge Challenge ↓ ↓ ↓ ↓↓ ↓ ODN ODN ↓ ↓ Day: 0 7 17 19 22 24 26 29 30 ↓ Endpoints

Results

Antigen challenge caused airway hyperreactivity. SEQ ID No. 313suppressed the development of antigen-induced airway hyperreactivity ina dose-related manner. The data is shown in FIGS. 31 and 32 as sampledose-response curves to methacholine to show effect of SEQ ID No. 313(1000 μg/kg). Dose-response curves to methacholine are quantified asarea under the curve. Results are mean±s.e.m. (n=6-8). * P<0.05 comparedwith antigen challenged, vehicle-treated group (Kruskal-Wallis multiplecomparison test).

An analysis of the full dose-response (RL) curves between the mice thatwere antigen challenged, treated with vehicle and each of the respectivemice that were antigen challenged, treated with SEQ ID No. 313 wascarried out using a repeated measures MANOVA. While there was asignificant difference (P<0.05) between the dose-response curves withthe 100 and 1000 ug/kg SEQ ID No. 313 treatment groups, there was nosignificant difference between the mice that were antigen challenged,treated with vehicle and the similarly treated animals dosed with 10ug/kg SEQ ID No. '313.

Example 27 In vivo Pharmacokinetics (PK) Study in the Rat

A PK study was carried out in rats to determine whether SEQ ID No. 313,a ‘semi-soft’ ODN, is cleared from plasma & tissues, particularly fromthe kidneys, at a faster rate than SEQ ID No. 329, a fullyphosphothioate ODN, which is identical in base sequence to SEQ ID No.313.

Methods

56 rats were administered by Intravenous (IV) & Intratratcheal (IT)routes 5 mg/kg (for both IV & IT) of SEQ ID No. 313 and SEQ ID No. 329.Plasma, Lungs, Kidneys were collected. The study lasted 5 days, with 14time points per dose group. 3 rats/time point for IV group (Total=42rats) and 4 rats/time point for IT group were used.

Results

FIG. 33 shows ODN concentrations in rat plasma following IV & ITadministration at 5 mg/kg. The plasma data shows that SEQ ID No. 313 iscleared more rapidly from plasma compared to SEQ ID No. 329 followingboth IV & IT administration.

FIG. 34 shows ODN concentrations in rat lungs following IV & ITadministration at 5 mg/kg. Following IV administration at the same doselevel, lung concentrations of SEQ ID No. 313 are lower than SEQ ID No.329 concentrations. After IT administration the difference is lessmarked. Lung data for SEQ ID No. 329 is only available up to 48 hrspost-date.

FIG. 35 shows ODN concentrations in rat kidneys following IV & ITadministration at 5 mg/kg. The kidney data indicates that absolutelevels of SEQ ID No. 313 in the kidneys are lower than corresponding SEQID No. 329 concentrations following both IV and IT administration. Therenal exposure to SEQ ID No. 313 after IT administration in particular,is markedly reduced compared to exposure to SEQ ID No. 329 at the samedose level. This can be seen more clearly in FIGS. 36 & 37.

FIG. 36 shows ODN concentrations in rat kidneys following IVadministration at 5 mg/kg. FIG. 37 shows ODN concentrations in ratkidneys following IT administration at 5 mg/kg, Following ITadministration both SEQ ID No. 313 & SEQ ID No. 329 are below the lowerlimit of quantitation (0.4-0.6 μg/g) in the kidneys for up to 1 hrpost-dose. After 1 hr, SEQ ID No. 329 can be detected in all kidneysamples collected during the study period (48 hrs). SEQ ID No. 313, onthe other hand, is only present in measurable levels for up to 7 hrspost-dose.

TABLE 15 Summary of mean PK parameters for SEQ ID No. 313 & SEQ ID No.329 following IV & IT administration to rats at a dose level of 5 mg/kg.Cmax Tmax T_(1/2) AUC_(0–INF) AUC_(0–48 h*) Dose Group Tissue ODN(ug/ml) (hrs) (hrs) (hr · ug/ml) (hr · ug/ml) IV Plasma 10 na na 0.209.5  9.3 (5 mg/kg) 17 na na 0.62 62.8  62.2 Lungs 10 1.4 0.25 0.17 0.470.35 17 14.4 0.083 2.5  23.7  20.8 Kidneys 10 6.6 0.083 24.9  184    12317 11.4 0.083 nc nc 346 IT Plasma 10 1.9 0.75 1.20 2.68 2.35 (5 mg/kg)17 2.1 2 2.3  9.01 7.46 Lungs 10 632 0.25 28.1  5540     5350 17 692 1(31)**   (7908)**    6505 Kidneys 10 0.49 2 7.8  5.81 2.34 17 3.8 7 ncnc 134 na—Not applicable nc—Not calculable. Could not be estimatedaccurately due to insufficient data points in terminal phase or terminalelimination phase not reached during the study period. *AUC_(0–48 h) orAUC_(0–LAST) when last measurable conc. before 48 h. **Very approximateestimate (based on 2 data points only in terminal phase). 10 - SEQ IDNo. 313 17 - SEQ ID No. 329

TABLES 16(a)–(c) Systemic and tissue exposure to SEQ ID No. 313 & SEQ IDNo. 329 following IT & IV administration to rats at a dose level of 5mg/kg. SEQ ID NO: 313: AUC_(0–48 hr) SEQ ID NO: 329 ODN Dose Route (hr ·ug/ml) Ratio (a) - Plasma data SEQ ID IT 2.35  0.32 (IT) No. 313 IV 9.30 0.15 (IV) IT:IV Ratio 0.25 SEQ ID IT 7.46 No. 329 IV 62.2 IT:IV Ratio0.12 (b) - Lung data SEQ ID IT 5350  0.82 (IT) No. 313 IV 0.35 0.017(IV) IT:IV Ratio 15286 SEQ ID IT 6505 No. 329 IV 21 IT:IV Ratio 313(c) - Kidney data SEQ ID IT 2.34 0.017 (IT) No. 313 IV 123  0.36 (IV)IT:IV Ratio 0.019 SEQ ID IT 134 No. 329 IV 346 IT:IV Ratio 0.39

Systemic and renal exposure of SEQ ID No. 313 was found to be markedlylower than exposure to SEQ ID No. 329 following administration of the 2ODNs by either the intravenous (IV) or intratracheal (IT) routes.

The plasma AUC for SEQ ID No. 313 after IT administration at 5 mg/kg was2.7 hr.μg/ml. The corresponding value for SEQ ID No. 329 was 9.0hr.μg/ml. Thus, the systemic exposure to SEQ ID No. 313 is a third ofthat seen with SEQ ID No. 329.

The kidney AUC for SEQ ID No. 313 after IT administration at 5 mg/kg was2.35 hr.μg/ml. The corresponding value for SEQ ID No. 329 was 134hr.μg/ml. Thus, for the same dose level, renal exposure to SEQ ID No.313 is only about 2% of the exposure seen with SEQ ID No. 329.

Unlike the case with plasma and kidneys, the lung exposure to SEQ ID No.313 following IT administration was not reduced to such a large extentwhen compared to the exposure to SEQ ID No. 329. The lung AUC for SEQ IDNo. 313 was approximately 70-80% of the lung AUC for SEQ ID No. 329 atthe same dose level. Since the lung is the target tissue, it isadvantageous that the clearance of the ODN from the lung is notincreased to the same extent as from plasma and kidneys.

FIG. 38 shows concentrations of SEQ ID No. 313 and its 8-mermetabolite(s) in rat kidneys following IV administration of SEQ ID No.313 at 5 mg/kg.

FIG. 39 shows concentrations of SEQ ID No. 313 and its 8-mermetabolite(s) in rat kidneys following IT administration of SEQ ID No.313 at 5 mg/kg. Due to methodological issues the data for the 8-mermetabolite of SEQ ID No. 313 in plasma & tissues is incomplete. However,8-mer data is available for some of the IV and all of the IT kidneysamples. This data shows that in most of those kidney samples where8-mer concentrations have been successfully measured, the levels of themetabolite exceed the levels of SEQ ID No. 313, indicating thatendonuclease activity is an important route of metabolism for SEQ ID No.313.

The introduction of a number of phosphodiester linkages (SEQ ID No. 313)into a fully phosphothioate backbone (SEQ ID No. 329) appears to haveincreased the degradation rate of the ODN, resulting in more rapidclearance, particularly from the kidneys.

Example 28 Activation of TLR9 Using Semi-Soft ODN Compared with FullyPhosphorothioate ODN

Methods

Stably transfected HEK293 cells expressing the human TLR9 were describedbefore [Bauer et al.; PNAS; 2001]. Briefly, HEK293 cells weretransfected by electroporation with vectors expressing the human TLR9and a 6×NFκB-luciferase reporter plasmid. Stable transfectants (3×10⁴cells/well) were incubated with ODN for 16 h at 37° C. in a humidifiedincubator. Each data point was done in triplicate. Cells were lysed andassayed for luciferase gene activity (using the Brightlite kit fromPerkin-Elmer, Ueberlingen, Germany). Stimulation indices were calculatedin reference to reporter gene activity of medium without addition ofODN.

Results

TLR9, is readily activated by ODNs containing optimal immunostimulatoryCpG sequences. We incubated a cell line stably expressing the human TLR9with a panel of semi-soft ODN and a panel of fully phosphorothioate ODNhaving the same ODN sequence as the semi-soft ODN. The results are shownin FIG. 40.

The results demonstrate that each of the semi-soft ODN shown in thetable below, SEQ ID NOs. 376, 378, 380, 382, 384, 241 activated higherlevels of TLR9 than the same sequence ODN having a fullyphosphorothioate backbone, SEQ ID NOs. 377, 379, 381, 383, 385, and 242respectively.

SEQ ID No. T*G*T*C_G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 376 SEQ ID No.T*G*T*C*G*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T 377 SEQ ID No.U*G*T*C_G*T*T*U*U*U*U*U*U*U*U*U*U*U*U*U 378 SEQ ID No.U*G*T*C*G*T*T*U*U*U*U*U*U*U*U*U*U*U*U*U 379 SEQ ID No.D*G*T*C_G*T*T*D*D*D*D*D*D*D*D*D*D*D*D*T 380 SEQ ID No.D*G*T*C*G*T*T*D*D*D*D*D*D*D*D*D*D*D*D*T 381 SEQ ID No.U*G*T*C_G*T*T*U*U*U*U*U_G_G_G_A_G_G*G*G 382 SEQ ID No.U*G*T*C*G*T*T*U*U*U*U*U*G*G*G*A*G*G*G*G 383 SEQ ID No.U*G*T*C_G*T*T*C*C*U*U*U_G_C_C_A_G_C*G*G 384 SEQ ID No.U*G*T*C*G*T*T*C*C*U*U*U*G*G*C*A*G*C*G*C 385 SEQ ID No.T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T*T*T*G*T*C_G*T*T 241 SEQ ID No.T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T 242

Example 29 Rp Internucleotide Linkages as Phosphodiester like Linkagesin Semi-Soft Oligonucleotides

Methods

Cell Culture Conditions and Reagents

For B cell proliferation assays, spleen cells from BALB/c mice (4-18weeks old) were cultured at 2-5×10⁵-10⁶ cells/ml in RPMI for 44 hr. in96-well microtiter plates, and then pulsed with 1 μCi of ³H thymidinefor 4-6 hr, before being harvested and cpm determined by scintillationcounting as previously described (Krieg et al., 1995). For Westernblots, WEHI-231 cells (American Type Culture Collection, Rockville, Md.)were cultured at 37° C. in a 5% CO₂ humidified incubator and maintainedin RPMI 1640 (Life Technologies, Gaithersburg, Md.) supplemented with10% heat-inactivated FCS (Life Technologies, Gaithersburg, Md.), 1.5 mML-glutamine, 50 μM 2-ME, 100 U/ml penicillin, and 100 μg/mlstreptomycin.

Oligonucleotides.

Oligodeoxynucleotides (PO-Oligos) and stereo-randomoligo(deoxynucleoside phosphorothioate)s [Mix-PS]-Oligos were purchasedfrom Operon Technologies (Alameda, Calif.) or prepared by the standardphosphoramidite method (Caruthers, 1985)(Stec et al., 1984). Theoligonucleotide [Mix-PS]-d(TCCATGACGTTCCTGACGTT) ([Mix-PS]-SEQ IDNO:386) was used as a positive control since it had previously beenfound to have strong immune stimulatory effects on mouse cells (Yi etal., 1996). For a CpG PS-Oligo with a minimal stimulatory motif, thesequence PS-d(TCAACGTT)-2066 was chosen for study as a typical CpG motifwith broad immune stimulatory effects representative of the broad familyof CpG DNA. This sequence was called [Mix-PS]-2066 when made with astereo-random backbone. When this octamer sequence was made with acomplete or partially stereo-defined backbone, the PS-Oligo was referredto as either [All-Rp-PS]-2066 or [All-Sp-PS]-2066 when the entirebackbone was stereo-defined, or as [CG-Rp-PS]-2066 or [CG-Sp-PS]-2066when only the CpG dinucleotide was stereo-defined. Other PS-Oligos usedincluded CpG PS-d(TCAACGTTGA) ([Mix-PS]-SEQ ID NO:387) and its All-Rp-and All-Sp-stereo-defined counterparts, and the control non-CpGPS-d(TCAAGCTTGA) [Mix-PS]-SEQ ID NO:388.

Stereo-defined phosphorothioate oligodeoxynucleotides were prepared bythe oxathiaphospholane method as described (Stec et al., 1995)(Stec etal., 1998). The syntheses were performed manually. The first nucleosideunits from the 3′-end were anchored to the solid support by aDBU-resistant sarcosinyl linker (Brown et al., 1989). Appropriatelyprotected deoxynucleosidyl monomers possessing3′-O-(2-thio-“spiro”-4,4-pentamethylene-1,3,2-oxathiaphospholane) moietywere synthesized and separated chromatographically into pureP-diastereomers. For synthesis of [CG-Rp-PS]-2066 and [CG-Sp-PS]-2066,unresolved mixtures of both P-diastereomers (in Rp:Sp ratio ca. 1:1)(Stec et al., 1998) were used for assembling of internucleotide linkagesof randomal configuration of P atoms. All synthesized oligomers werepurified by two-step RP-HPLC: DMT-on (retention times in the range 23-24minutes) and DMT-off (retention times 14-16 minutes); chromatographicsystem: an ODS Hypersil column, 5 μm, 240×4.6 mm, 0-40% CH₃CN in 0.1 Mtriethylammonium bicarbonate, pH 7.5, gradient 1%/min. Their purity wasassessed by polyacrylamide gel electrophoresis.

For studies of PS-Oligo uptake, fluorescein conjugated stereoregularPS-Oligos were prepared by solid phase elongation of manuallysynthesized stereo-defined PS-oligomers. After detritylation step,fluorescein phosphoramidite (ChemGenes Corporation, Ashland, Mass.;working concentration 125 mg/mL) and 1-H-tetrazole were routinely added(coupling time 120 s), followed by sulfurization with S-Tetra reagent(Stec et al., 1993). Cleavage from the support and deprotection wereperformed with conc. ammonium hydroxide for 1 h at room temperature and4 h at 55° C., respectively. The resulting oligomers were purified byone step RP-HPLC (vide supra). Because of remarkable hydrophobicity offluoresceine moiety, the Rp- and Sp-oligomer was eluted at retentiontimes 14.5, 14.8 and 14.7, 15.0 min, respectively, i.e. at the end offailed sequences. In both cases two P-diastereomers were eluted due tonon-stereospecificity of the phosphoramidite/sulfurization method ofelongation with the fluorescein monomer.

Western Blot Analysis

Cells were harvested and resuspended in fresh medium at a concentrationof 2*10⁶ cells/ml. Cells were allowed to rest for four hours prior to a40-minute stimulation. Cells were harvested and washed three times withcold PBS. Cells were lysed in 0.05M Tris (pH 7.4), 0.14M NaCl, 1% NP-40,0.001M Na₃VO4, 0.0 M NaF, 4.3 mg/ml 1-glycerophosphate, 0.002M DTT, 50μg/ml PMSF, 12.5 μg/ml antipain, 12.5 μg/ml aprotinin, 12.5 μg/mlleupeptin, 1.25 μg/ml pepstatin, 19 μg/ml bestatin, 10 μg/mlphosphoramidon, 12.5 μg/ml trypsin inhibitor by freezing and thawingfollowed by a 30 minute incubation on ice. The samples were thencentrifuged at 10,000×g for 10 min at 4° C. The supernatants were savedas whole cell lysates for further analysis. Equal amounts of whole celllysates (20 μg) were boiled in SDS sample buffer for 5 minutes beforebeing subjected to electrophoresis on an 11% denaturing polyacrylamidegel. After electrophoresis, proteins were transferred to nitrocellulosemembranes using a semi-dry blotter (Bio-Rad Laboratories, Hercules,Calif.). Blots were blocked with 5% non-fat milk before hybridizationwith phospho-SAPK/JNK (Cell Signaling Technology, Beverly, Mass.), IκB-αand JNK1 (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.). Blotswere visualized using enhanced chemiluminescence reagents (ECL, AmershamInternational) according to the manufacturer's protocol.

Results

Induction of spleen cell ³H thymidine incorporation by the Spstereoisomer of CpG PS-Oligos. In order to determine thestereo-specificity of the immune stimulatory effects of CpG DNA, BALB/cspleen cells were cultured with stereo-defined octanucleotidesPS-d(TCAACGTT)-2066 in which all of the internucleotide linkages areeither R_(p) or S_(p) configuration at the concentrations indicated inTable 17. The cells were cultured for 48 hr, which allows sufficienttime for B cells to be induced to proliferate by the CpG motifs (Krieget al., 1995). The stereo-random [Mix-PS]-2066, possessing a CpG motif,induced strong dose-dependent spleen cell proliferation (Table 17). TheSp isomer also induced proliferation, and appeared to be marginally morepotent than [Mix-PS]-2066. In contrast, the Rp stereoisomer did notinduce any detectable proliferation, which was consistent with thefindings of Yu et al., (Yu et al., 2000).

TABLE 17 Induction of spleen cell proliferation by the Sp stereoisomerof CpG octamers. Oligo Concentration cpm SI None 2170 1 (medium) 2066(stereo-random CpG) 0.4 μM 3154 1.5 ″ 2.4 μM 16,525 7.6 ″ 4.8 μM 30,81114.2 Rp (2066) 0.4 μM 1207 0.6 ″ 2.4 μM 985 0.5 ″ 4.8 μM 640 0.3 Sp(2066) 0.4 μM 9567 4.4 ″ 2.4 μM 35,372 16.3 ″ 4.8 μM 43,591 20.1 Rp(2066) + 2066¹ 0.4 μM 1,597 0.7 ″ 2.4 μM 10,255 4.7 ″ 4.8 μM 15,841 7.3SI = stimulation index compared to medium control ¹each of the twoPS-Oligos were added to the indicated concentration at the start ofculture

Our previous studies had demonstrated that decamer CpG PS-Oligos haveimproved immune stimulatory effects compared to the octamers used in thefirst experiments. Therefore, these experiments were repeated using theconstruct PS-SEQ ID NO:387, which was synthesized either as astereo-random [Mix-PS]-SEQ ID NO:387, or in the All-Rp- or All-Sp-form.Again, both the [Mix-PS]-SEQ ID NO:387 and the [All-Sp-PS]-SEQ ID NO:387induced strong 3H thymidine incorporation in a dose dependent manner.However, in this case, the [All-Rp-PS]-SEQ ID NO:387 was also able toinduce a substantial increase in cell proliferation at the highestconcentrations, indicating that it retained at least partial stimulatoryactivity.

Preference for Rp chirality at the CpG dinucleotide in octamerPS-Oligos. It remained unclear whether the apparent preference for theSp stereoisomer in the initial experiments resulted from an effectwithin the CG dinucleotide itself, or whether this effect may be outsidethe CG. In order to determine this, two octamers PS-2066 weresynthesized in which the backbone was stereo-random except for thelinkage between the central CG, which was defined as either Sp or Rp.Surprisingly, this experiment appeared to give the opposite result fromthose using PS-Oligos in which the entire backbone was stereoregularsince [CG-Rp-PS]-2066 caused as strong an increase in spleen cell ³Hthymidine incorporation as the control stereorandom PS-Oligo Incontrast, PS-Oligo [CG-Sp-PS]-2066 was essentially inactive.

Inhibition of spleen cell ³H thymidine incorporation by the Rstereoisomer of CpG PS-Oligo. The level of 3H thymidine incorporation inthe wells treated with the Rp stereoisomer was lower than the controlwells, suggesting possible inhibitory activity, although no cytotoxicitywas apparent on microscopic examination of the cells. Indeed, when cellswere cultured with an equimolar mixture of the [Mix-PS]-2066 and theAll-Rp stereoisomer, there was an approximate 50% reduction in the levelof ³H thymidine incorporation compared to cells cultured with only the[Mix-PS]-2066 (Table 17).

Preferential immune stimulation by [Rp-PS]-Oligos at early timepoints.The ³H thymidine incorporation assays performed in the precedingexperiments are vulnerable to an artifact resulting from PS-Oligodegradation, with release of cold thyinidine that competes with thelabeled material, artificially suppressing its incorporation (Matson etal., 1992). Previous studies have demonstrated that [Rp-PS]-Oligos arefar more susceptible to nuclease degradation than Sp counterparts. Thus,it was possible that the apparent lack of stimulatory effect of the[Rp-PS]-Oligo in our 3H thymidine incorporation assays may have been amisleading artifact that did not reflect the true effects of the[Rp-PS]-Oligo. In order to detect the stimulatory effects of the[Rp-PS]-Oligo at an early timepoint, before the PS-Oligo can bedegraded, and as an independent biologic assay for CpG-inducedstimulation, we tested the ability of these PS-Oligos to induce rapidphosphorylation of the regulatory mitogen activated protein kinase, JNK.Surprisingly, we found that upon treatment with CpG sequences PS-SEQ IDNO:386 and PS-SEQ ID NO:387, within forty minutes JNK phosphorylationwas induced strongly not by the [Sp-PS]-isomers but only by thestereorandom [Mix-PS]- and by [Rp-PS]-isomers. A control non-CpG[Mix-PS]-SEQ ID NO:388 did not induce detectable JNK phosphorylation.All samples contained comparable amounts of total JNK protein.

Although no effect of the CpG [Sp-PS]-Oligo could be detected in the JNKphosphorylation assay, the oligo was biologically active in thisexperiment, because the level of the inhibitory protein IκB-α wasreduced by all of the CpG PS-Oligos, regardless of stereoisomer, but notby the control non-CpG PS-SEQ ID NO:388.

Stereo-independence of PS-Oligo cell surface binding and uptake. Onepotential explanation that could contribute to the observed differencesin bioactivity of the PS-Oligo stereoisomers is that cell binding oruptake of the PS-Oligos may be stereo-dependent. To test thispossibility, P-stereo-defined PS-Oligos were synthesized withfluorescent tags and incubated with cells. Consistent with the resultsof past studies, the PS-Oligos showed a concentration-dependent andtemperature-dependent pattern of cell uptake. Notably, there was nodetectable difference in the binding or uptake of the Rp or SpPS-Oligos.

Example 30 Semi-soft C Class Oligonucleotide ODN 316 and Semi-soft BClass Oligonucleotide ODN 313 Reduce Antigen-induced AirwaysInflammation in vivo

This study assessed the in vivo effect of ODN 316 in a murine model ofantigen-induced airways inflammation. The B class ODN 313 was includedin the study for comparison.

Methods. Mice (male BALB/c) were sensitized on study days 0 and 7 withantigen (ovalbumin, 10 μg, i.p.) with aluminum hydroxide adjuvant(Pierce Alum).

Mice were antigen challenged by exposure to inhaled ovalbumin aerosol,twice each week for two consecutive weeks. The first challenge wasadministered on study day 21. The aerosol was generated for 1 hour froma 1% solution of ovalbumin in PBS using a DeVilbiss Ultraneb nebulizer.Separate mice acted as unchallenged controls.

ODN 316 or ODN 313 (1-100 μg/kg) or vehicle (saline, 20 μl) wereadministered intranasally once each week, two days before the firstantigen challenge of the week.

Endpoints were assessed on study day 33 (i.e., 48 hours after the lastantigen challenge). Cells in airways were recovered by bronchoalveolarlavage. Differential cell counts were made by an Advia automated cellcounter with random samples checked by visually counting cells oncytocentrifuge preparations stained with Wright-Giemsa stain. Numbers ofCD4⁺ T cells (CD3⁺CD4⁺ cells) were counted by flow cytometry. Resultswere expressed in terms of mean±SEM for each group. Significance wasmeasured using the Kruskall-Wallis multiple comparison test.

Results. Antigen challenge caused an increase in the total number ofleukocytes in the airway lumen. This increase was predominantly due toan accumulation of eosinophils (e.g., 3×10⁵ eosinophils/ml inantigen-challenged, vehicle-treated mice versus <1×10⁴ eosinophils/ml inunchallenged mice). The eosinophilia was suppressed significantly by ODN316 or ODN 313 (e.g., ca. 5×10⁴ eosinophils/ml (P<0.05) inantigen-challenged mice treated with 100 μg/ml of either ODN).

Antigen challenge also caused an accumulation of CD4⁺ T cells that wassignificantly suppressed by either ODN (e.g., ca. 2×10⁴ CD4⁺ T cells/mlin antigen-challenged mice treated with 100 μg/ml of either ODN, versusca. 1.3×10⁵ CD4⁺ T cells/ml (P<0.05) in antigen-challenged,vehicle-treated mice).

Conclusions. Each of semi-soft C class ODN 316 and semi-soft B classoligonucleotide ODN 313 suppressed antigen-induced airways eosinophiliaand the accumulation of CD4⁺ T cells in vivo.

Example 31 Comparison of Semi-soft B, C, and T Class ODN: Induction ofCytokine Secretion from Murine Splenocytes in vitro

This study investigated the ability of semi-soft B, C, and T class ODNsto induce cytokine secretion from murine splenocytes in vitro.

Methods. Splenocytes from BALB/c mice were harvested and pooled.Splenocytes were incubated in 48-well culture plates at 1×10⁷ cells/1 mlin RPMI 1640+10% fetal bovine serum containing individual ODN (0, 0.001,0.01, 0.1, 1 or 10 μg/ml). Tested ODN included semi-soft B class ODN20674, semi-soft C class ODN 316 and ODN 317, and semi-soft T class ODN319 and ODN 320.

After 48 hours incubation (37° C., 5% CO₂), culture medium was removedand concentrations of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, GM-CSF,IFN-γ and TNF-α were measured using the Luminex cytokine multiplexsystem. IL-12p40, IFN-α and IP-10 concentrations were measured by ELISA.Lower limits of accurate detectability were 3.2-10 pg/ml. Activationstatus of cells was assessed by measuring CD40, CD69 and CD86 expressionon CD3+ and B220+ cells by flow cytometry.

Results. Each of the ODNs induced activation of B cells (B220+ cells) asmeasured by increased expression of CD40, CD69 and CD86, and activationof T cells (CD3+ cells) as measured by increased expression of CD69.

The ODNs induced secretion of IL-6, IL-10, IL-12p40, IFN-α, TNF-α andIP-10. Titers of the other cytokines measured were not increased. Forexample, at an ODN concentration of 1 μg/ml, cytokine secretion levelswere found to be as follows (all expressed in pg/ml):

TABLE 18 In Vitro Cytokine Secretion in Response to Semi-Soft B, C, andT Class ODN ODN IL-6 IL-10 IL-12p40 IFN-α TNF-α IP-10 313 4000 410 30012 150 400 316 3600 820 820 90 400 780 317 2200 410 790 140  340 760 3191200 200 300 nd  50  30 320  150 nd 160 nd  15  25 nd—not detected

When compared with the semi-soft B class ODN 313, the two semi-soft Cclass ODNs induced higher titers of IL-10, IL-12p40, IFN-α, TNF-α andIP-10, but did not cause more marked B cell activation. The twosemi-soft T class ODNs appeared to be less effective than the semi-softB and C class ODNs as cytokine inducers.

Conclusions. Each B class and C class ODN induced a profile of cytokineinduction that was consistent with activation of TLR9, and each causedactivation of B cells. The T class ODNs were less effective cytokineinducers.

When compared with the semi-soft B class ODN 313, the semi-soft C classODNs 316 and 317 each induced higher concentrations of immune-modifyingcytokines, but without inducing more B cell activation. This datasuggests a therapeutic benefit of the C class ODNs.

Example 32 Cytokine, Antibody, and CTL Induction in vivo in Response toCpG ODN

Cytokine measurements: BALB/c mice were administrated 400 mg ODN (SEQ IDNO.s 294 (soft), 241 (semi-soft), 242, and 286) by SC injection. Animalswere bled at 3 hours post injection and IP-10, IFN-gamma and TNF-alphalevels in plasma was measured by ELISA. The Results are shown in Theresults are shown in FIGS. 41A & B (IP-10), C (IFN), and D & E (TNF)

Antibody Response: BALB/c mice were immunized with 1 mg HBsAg alone orin combination with CpG ODN by IM injection. Animals were boosted at 4weeks post primary immunization. Antibody titers were measured by endpoint ELISA. IgG isotype titers were measured at 2 weeks post boost byend point ELISA. The results are shown in FIGS. 42A and B.

Cytotoxic T lymphocyte Response: BALB/c mice were immunized with 1 mgHBsAg alone or in combination with CpG ODN by IM injection. Animals wereboosted at 4 weeks post primary immunization. CTL activity was measuredat 4 wk post boost by 51Cr release assay. The results are shown in FIG.42C.

Thus, soft and semi-soft ODN have similar or are better in activatingmurine immune system as seen by both in vitro and in vivo studies andcan augment antigen specific immune responses

Example 33 Use of CpG ODN in in vivo Anti-cancer Therapy

The ODN of the invention were tested for efficacy in three cancer modelsas mono-therapies. Initially the ODN were administered to mice havingrenal cell carcinoma (renca). The methods were performed as follows:Tumors were induced by injecting 2×105 renca cells SC in the left flankof mice on Day 0. Treatment followed an involved SC injections of PBS,CpG ODN 241 or 242 weekly for 5 weeks starting on day 10 post tumor cellinjection. The results are shown in FIGS. 43A and B.

The second model tested was murine non-small cell lung cancer (Lewislung carcinoma). Tumors were induced by injecting 2×10⁶ Lewis LungCarcinoma cells SC in the left flank of mice on day 0. Treatmentfollowed and involved SC injections of PBS, 100 mg CpG ODN 241 or 242 ondays 1, 3, 7 & weekly for 2 months. The results are shown in FIGS. 43Eand F.

A third model tested was murine neuroblastoma. 1×106 Neuro2a cells wereinjected SC in the left flank on day 0. SC injections of PBS, 100 mg CpGODN 241 or 242 were performed daily from day 10 to day 15. The resultsare shown in FIGS. 43C and D.

Thus, semi-soft ODN can control growth of cancer (murine renca, LLC,neuroblastoma) and enhance survival of mice bearing these cancers

Example 34 Peri-renal Inflammation Resulting from Administration ofSoft, Semi-soft and Hard ODN in BALB/c Mice in TLR-9 Knockout Mice

Peri-renal inflammation was assessed in BALB/c mice in TLR-9 knockoutmice. The results are shown in Table 19 and 20 respectively. Semi softODN (241) induced less inflammation at the site of injection, induced no(100 mg dose) or little (250 mg dose) peri-renal inflammation, and werebetter tolerated following multiple administrations of ODN

TABLE 19 Kidney Renal capsule Adipose tissue parenchyma granulomatousgranulomatous Group inflammation inflammation inflammation PBS NormalNormal Normal 5/5 5/5 5/5 242 Mild Mild to moderate Mild to moderate 100mg 2/5 5/5 4/5 242 Mild Mild to moderate Marked 250 mg 1/4 4/4 4/4 241Normal Normal Normal 100 mg 5/5 5/5 5/5 241 Mild Mild Mild to moderate250 mg 2/5 2/5 3/5

TABLE 20 Kidney Renal capsule Adipose tissue parenchyma granulomatousgranulomatous Group inflammation inflammation inflammation PBS NormalNormal Normal 5/5 5/5 5/5 242 Normal Normal Normal 100 mg 5/5 5/5 5/5242 Normal Normal Normal 250 mg 5/5 5/5 5/5 241 Normal Normal Normal 1005/5 5/5 5/5 241 Normal Normal Normal 250 mg 5/5 5/5 5/5

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

1. An oligonucleotide having the following structure: (SEQ ID NO: 313)5′ T*C_G*T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C_G*T* T  3′,

wherein * refers to the presence of a phosphorothioate internucleotidelinkage, and wherein _ refers to the presence of a phosphodiesterinternucleotide linkage and wherein the oligonucleotide has a length of24-40 nucleotides.
 2. The oligonucleotide of claim 1, wherein theoligonucleotide consists essentially of 5′ (SEQ ID NO: 313) 5′T*C_G*T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C_G*T* T 3′.


3. The oligonucleotide of claim 1, wherein the oligonucleotide consistsof (SEQ ID NO: 313) 5′ T*C_G*T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C_G*T*T 3′.


4. An oligonucleotide having the following structure: (SEQ ID NO: 313)5′ T*C_G*T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C_G*T*  T 3′

wherein each * refers to a phosphorothioate internucleotide linkage andeach _ refers to a phosphodiester internucleotide linkage, and whereinthe oligonucleotide is 24 nucleotides in length.
 5. A pharmaceuticalcomposition comprising an oligonucleotide as defined in claim 1 and apharmaceutically acceptable carrier.
 6. A pharmaceutical compositioncomprising an oligonucleotide as defined in claim 4 and apharmaceutically acceptable carrier.
 7. An immunostimulatoryoligonucleotide having at least two internal cytosine-guanine (CG)dinucleotides, wherein C is unmethylated cytidine, and G is guanosine,wherein the at least two internal CG dinucleotides have phosphodiesterinternucleotide linkages, wherein optionally each additional internal CGdinucleotide has a phosphodiester or stabilized internucleotide linkage,wherein all other internucleotide linkages are stabilized with aphosphorothioate internucleotide linkage, and wherein theimmunostimulatory oligonucleotide is 6-100 nucleotides long.
 8. Theoligonucleotide of claim 7, wherein the immunostimulatoryoligonucleotide comprises a plurality of internal CG dinucleotideshaving a phosphodiester internucleotide linkage.
 9. The oligonucleotideof claim 7, wherein the immunostimulatory oligonucleotide is a B-Classimmunostimulatory oligonucleotide.
 10. The oligonucleotide of claim 7,wherein the immunostimulatory oligonucleotide is not an antisenseoligonucleotide, triple-helix-forming oligonucleotide, or ribozyme. 11.The oligonucleotide of claim 7, wherein the oligonucleotide has abackbone comprising deoxyribose or ribose.